Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine (C) or adenine (A), but no method for guanine (G) or thymine (T) editing is available at present. Here we developed a deaminase-free glycosylase-based guanine base editor (gGBE) with G editing ability, by fusing Cas9 nickase with engineered N-methylpurine DNA glycosylase protein (MPG). By several rounds of MPG mutagenesis via unbiased and rational screening using an intron-split EGFP reporter, we demonstrated that gGBE with engineered MPG could increase G editing efficiency by more than 1500 fold. Furthermore, this gGBE exhibited high base editing efficiency (up to 81.2%) and high G-to-T or G-to-C (i.e. G-to-Y) conversion ratio (up to 0.95) in both cultured human cells and mouse embryos. Thus, we have provided a proof-of-concept of a new base editing approach by endowing the engineered DNA glycosylase the capability to selectively excise a new type of substrate. Base editing is a powerful technology for basic research and therapeutic applications ,2. CurreBase excision proteins in base excision repair (BER) pathways have been successfully harnessed for developing new base editing tools ,15. The 6-ethenoadenine (ϵA) and deaminated adenine hypoxanthine (Hx) [in vitro [There are many types of DNA glycosylases, including N-methylpurine DNA glycosylase (MPG), 8-oxoguanine DNA glycosylase (OGG1) and mutY DNA glycosylase (MUTYH), that could excise various damaged purine bases ,21–23. Tine (Hx) . Althougin vitro , overexpin vitro . This imHere, we developed a novel tool, namely glycosylase-based guanine base editor (gGBE), to achieve high editing efficiency for G-to-C and G-to-T conversion in mammalian cells. After four rounds of mutagenesis, we obtained marked enhancement of G base editing activity, as compared with that obtained by using wild-type (WT) MPG. We characterized the editing profile of gGBE by targeting dozens of endogenous genomic loci in cultured mammalian cells as well as mouse embryos, demonstrating its high G-to-Y (Y = C or T) base editing efficiency. Thus, DNA glycosylase could be engineered into a new base editing tool.We have attempted to develop a novel base editing system by harnessing the BER pathway triggered by depurination of G or A. By fusing human MPG at the C-terminus of Cas9 D10A nickase (nCas9), we generated a prototype version of deaminase-free glycosylase-based base editor (gBE), in which MPG may remove normal G or A, two purines with similar structure of Hx, to generate AP sites, with nCas9 nicking the opposite non-edited strand. This allowed the damaged DNA to preferentially use the edited strand as a template for DNA repair and/or DNA replication. Therefore, nucleotide incorporation opposite the AP site via TLS would lead to diverse editing outcomes Fig. . To convTo further increase the G-to-T activity of gGBEv3, we performed protein engineering by several rounds of rational mutagenesis of the MPG moiety, using the G-to-T reporter to evaluate the editing activity Fig. . Based oWe then performed another round of mutagenesis and screening based on gGBEv4 to further elevate G editing activity of gGBE. We mutated the R163–V179 region of MPGv4 by sequential replacement with amino acids of distinct properties, including glutamic acid (with negative charged side chain), valine , glycine (with no side chain), or tyrosine (with large hydrophobic side chain) . Although most of these mutations reduced the G editing activity, we found three variants, gGBEv4.1 (v4 with I170V), gGBEv4.2 (v4 with S169G) and gGBEv4.3 (v4 with R163Y), that showed elevated editing efficiency by 1.06-, 1.28- and 1.09-fold, respectively , as comp+ cells were FACS-sorted for target deep sequencing analysis. We obtained a gradual elevation of overall G editing efficiency at G7 from 6.4% to 78.5% for site 3, and from 7.5% to 80.3% for site 10, respectively , and mCherryely Fig. . The G-tWe further characterized the editing profiles of gGBEv6.3 by targeting 24 endogenous genomic loci, most of which were used in previous base editing studies ,35,36. Windependent off-target DNA editing using orthogonal R-loop assay in five dSaCas9 R-loops, reported in previous studies [We have analyzed the guide-dependent off-target activity of gGBEv6.3 at several previously reported ,35 and i studies ,35. We f studies ,35. MoreDMD (Duchenne muscular dystrophy) exon 45 with gGBEv6.3, and achieved a high efficiency of G editing (up to 30.3%) with a high G-to-Y ratio (up to 0.88) when targeting DMD site 1 , as well as editing that bypasses PTCs Fig. . The inae 1 Fig. .Tyr  gene with gGBEv6.3 for creating PTCs , with 20 mouse embryos used for each of three Tyr-targeting sgRNAs. We found highly efficient G editing , ABE and CBE, as well as their derivatives (such as AYBE and CGBE), perform base editing with deamination of A or C as the first key step . In thisIn all human pathogenic SNPs , there are about 10% C-to-G and 5% T-to-G SNPs . AlthougAlthough we have found a low off-target effect of the gGBE editor on several targeted genes in culture mammalian cells, a comprehensive evaluation of gGBEs through high-throughput whole-genome sequencing methods, such as GOTI ,39, SAFEIn summary, we have shown that DNA glycosylases could be engineered into proteins that selectively excise a specific nucleotide base. Thus, besides G base editing illustrated here, glycosylase-based base editor could also be developed by similar protein engineering methods for base editing of A, C and T, providing a complete set of base editing tools for gene editing studies.Base editor constructs used in this study were cloned into a mammalian expression plasmid backbone under the control of a EF1α promoter by standard molecular cloning techniques. Intron-split EGFP reporters were engineered as previously described . In brieTarget sequencing data analysis has been previously described . In briehttps://www.addgene.org/Huawei_Tong/ (Addgene plasmid nos. 202629-202630). All data are available in the main text or the https://www.ncbi.nlm.nih.gov/bioproject/PRJNA971099. All relevant original data are available from the corresponding authors upon request.Expression plasmids used in this study have been deposited at Addgene and will be available at nwad143_Supplemental_FilesClick here for additional data file.


The smallest arenavirus gene product, Z protein, plays critical roles in the virus life cycle. Z is the major driving force of budding and particle production because of a unique property that defines self-assembly. In addition to the roles in budding, Z also participates in the suppression of type I interferon production to evade host antiviral immunity. Therefore, Z and its assembled form are an attractive drug target for arenaviral hemorrhagic fever, such as Lassa fever. Here, we developed a biosensor that enabled the evaluation of the prototype arenavirus, lymphocytic choriomeningitis virus (LCMV), Z assembly using the principle of Förster resonance energy transfer (FRET). This FRET biosensor consisted of three tandem Z that were sandwiched between super-enhanced cyan-emitting fluorescent protein and variant of a yellow-emitting mutant of green fluorescent protein so that Z-Z intermolecular binding via the really interesting new gene finger domain increased the emission ratio. To identify novel anti-arenavirus compounds, the FRET biosensor was employed to screen the PathogenBox400 for inhibitors of Z assembly in a 96-well plate format. The assay performed well, with a Z’-factor of 0.89, and identified two compounds that decreased the emission ratio of the FRET biosensor in a dose-dependent manner. Of them, the compound, 5,6,7,8-tetrahydro-7-(benzyl)-pyridothienopyrimidin-2,4-diamine, was found to significantly inhibit LCMV propagation in infected cells. Thereby, the present study demonstrated that a novel FRET biosensor incorporating Z assembly built on FRET and named Zabton, was a valuable screening tool to identify anti-arenavirus compounds in the context of inhibition of Z assembly. Several arenaviruses are responsible for severe hemorrhagic fever (HF) in humans, such as Lassa fever (LF) and the South American HF . In addiAll arenaviruses encode five mature products: a nucleoprotein (NP), an RNA-dependent RNA polymerase (RdRp or L), the matrix protein (Z), and two envelope glycoproteins (GP1 and GP2) . The glyHere, we developed a novel FRET-based biosensor to monitor the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV)-Z protein assembly in both living cells and cell lysates. The biosensor encompassed unique properties of Z protein, including zinc-binding, self-assembly, and suitability for high-throughput screening. Preliminary screening with four hundred compounds using the FRET biosensor worked well and acquired potential hit compounds. Through a typical bioassay and an LCMV infection assay, one compound was finally identified, 5,6,7,8-tetrahydro-7-(benzyl)-pyridothienopyrimidin-2,4-diamine, as a useful candidate for inhibiting arenavirus propagation.E. coli , as reported for Lassa virus Z protein . HeLa and HEK293T cells were obtained from RIKEN and maintained in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% (v/v) fetal bovine serum . Vero 76 cells were obtained from the Health Science Research Resources Bank  and were maintained in DMEM supplemented with 1% penicillin/streptomycin and 10% FBS. The Medicines for Malaria Venture (MMV) Pathogen Box contained 400 compounds as 10 mM stock solutions in dimethyl sulfoxide (DMSO), and was obtained free of charge from MMV (https://www.mmv.org). N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) was purchased from Dojindo . Ethylenediaminetetraacetic acid (EDTA) was purchased from Nacalai Tesque Inc. . Rabbit antisera against LCMV Z protein was produced by immunizing rabbits with recombinant LCMV Z protein produced in Expression vectors for Venus, a variant of a yellow-emitting mutant of green fluorescent protein, and SECFP, a super-enhanced cyan-emitting fluorescent protein, were kindly provided by A. Miyawaki (RIKEN). Zabton was generated using the pCAGGS eukaryotic expression vector; it encoded a chimeric protein that consisted of Venus, the 5× GS linker (Gly-Gly-Gly-Gly-Ser), multiple LCMV-Z proteins, the 5× GS linker, and SECFP from the amino terminus see B. The coC7H1/A8 expression vectors using the ExpiFectamine 293 transfection reagent (Life Technologies), according to the manufacturer’s protocol. At 72 hours post-transfection, cells were harvested by centrifugation, lysed in lysis buffer , clarified by additional centrifugation, and subjected to spectroscopy, as well as immunoblotting. Gene transfer into HeLa cells was performed using Lipofectamine 3000 transfection reagents .Expi293 cells were transfected with Zabton or ZabtonThe fluorescence of Zabton probes expressing cell lysates were measured using a SpectraMax i3x multimode microplate reader  or an ARVO-X3 . The fluorescence emissions of SECFP (480 nm±10 nm) and FRET (530 nm±10 nm) at an excitation wavelength of 420 nm±10 nm were measured with a SpectraMax i3x multimode microplate reader. With an ARVO-X3 system, the fluorescence emissions of SECFP (460 nm) and FRET (535 nm) at an excitation wavelength of 406 nm were measured. After background subtraction, the emission ratio of FRET and SECFP (FRET/CFP) was calculated. A fluorescence spectrum was obtained by means of an FP-6500 fluorescence spectrometer (JASCO Co.), with an excitation wavelength of 420 nm. For the demonstration of FRET, the cell lysates were incubated with 50 μg/ml proteinase K  at 37°C for 30 min and analyzed with the fluorescence microplate reader, or the spectrometer. Fluorescence measurement of the probes was performed with a SpectraMax i3x multimode microplate reader unless otherwise indicated.C7H1/A8 and Zabton-expressing samples as negative (0%) and positive (100%) controls, respectively. Z’=1–{(3·Standard deviation (S.D.)100%+3·S.D.0%)/(Mean100%–Mean0%)}. S/B=Mean100%/Mean0%. S/N=(Mean100%–Mean0%)/S.D.0%The 400 compounds of the Pathogen Box were prepared according to the MMV foundation instructions, and screened on 96-well plates  using Zabton-expressing Expi293 cell lysates. The lysates (50 μL) were arrayed into 96-well plates, and 50 μL of the working stock  with DMSO) was added to each well of the 96-well plate, resulting in a final screening concentration of 10 μM. DMSO (0.05%) was used as a negative control. The fluorescence emissions of SECFP and FRET were obtained by means of a SpectraMax i3x multimode microplate reader. The quality of the screen was evaluated using the Z’-factor, signal to background (S/B) and signal to noise (S/N) based on the difference in the emission ratios between Zabton2 control) and plated on a collagen-coated glass base plate . Cell image acquisition was performed, as previously described (HeLa cells expressing Zabton were cultured in phenol red-free DMEM (Invitrogen) buffered with 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) , incubated at 95°C for 5 min, and then, the samples were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The separated proteins were transferred to a polyvinylidene difluoride membrane  and subjected to immunoblot analysis using antibodies indicated in the figure legends. Proteins were detected using the Enhanced Chemiluminscence (ECL) Western Blotting Detection Reagent  and a LAS-3000 UV mini image analyzer , as previously described (4 cells) were seeded into a 96-well plate one day before infection. The cells were infected with 1:10 serial dilutions of the viruses, and incubation was continued for 16 hours at 37°C and 5% CO2. The cells were then fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature and incubated with phosphate-buffered saline (PBS) containing 0.1% Tween 20 for 1 hour at room temperature. Blocking with 10% FBS in dilution buffer  was performed at 4°C overnight. Fixed cells were stained with anti-LCMV NP antibody and fluorescein isothiocyanate-conjugated anti-rat IgG antibody for 3 hours each at room temperature. The LCMV NP-positive cells were visualized using an AxioVert A1 microscope , and used to determine the fluorescent focus unit/mL, as the virus titer.An immunofocus assay was performed to determine LCMV titers. Vero 76 cells (3×10The Armstrong strain of recombinant LCMV (rLCMV) was rescued using the reverse genetics method and propagated in Vero 76 cells (4 cells/well) and treated with increasing concentrations of compounds . At 24 h post-treatment, cells were incubated with the CellTiter-Glo reagent, and the assay was performed according to the manufacturer’s recommendations.The effect of hit compounds on cell viability was determined using the CellTiter-Glo Luminescent Cell Viability assay . Briefly, HEK293T cells were seeded into 96-well plates , followed by Dunnett’s multiple comparisons test. Welch’s t-test was used as a comparison between two groups.Excel and GraphPad Prism 5  software were used for all statistical analyses. Quantitative data are presented as the mean ±S.D. from at least three independent experiments (unless otherwise indicated). For all calculations, C7H1/A8. The emission ratio of Zabton-3C7H1/A8 was significantly reduced compared to that of the wild-type (WT) Zabton-3  were comparable to that of WT-Zabton (53.1±0.4%)  B. Additi.1±0.4%)  and 2. U.1±0.4%)  and 2. WNext, we evaluated the effect of the hit compounds from the FRET-based screening assay on LCMV propagation. While the treatment of compound #1 on LCMV-infected cells did not show any inhibitory effect of virus propagation, the production of infectious viruses from compound #4 treated LCMV-infected cells (both 20 and 50 μM) was reduced compared to that of DMSO-treated LCMV-infected cells A. Since Novel screening of 10 μM compounds using a FRET biosensor named Zabton identified two hits after a cut-off of three SDs from the average of the negative control and the exclusion of compounds through a validation assay. Out of the two selected compounds, one compound #4, -pyridothienopyrimidin-2,4-diamine) exhibited inhibitory activity against LCMV propagation at 20 μM. In the present study, we thus demonstrated that the screening method using the novel FRET sensor Zabton was a promising platform for the identification of effective drugs against arenavirus replication and propagation.Since the arenaviral Z protein is not only a structural component of the virus, but also interacts with cellular proteins to disorder the host immune system, the Z protein is an attractive drug target for arenavirus infectious diseases . Of noteZabton-based screening identified two-hit compounds (#1 and #4), but the virus propagation was only inhibited by compound #4, not by #1. Although further experiment will be required to clarify the different outcome, there are several possibilities which might explain this controversial result. Arenaviral Z protein has been known to interact with the other viral components, including NP, L, and GPC, during the virus infection (The elegant crystal structure of the Lassa virus Z protein provides evidence that the dodecameric form is built based on dimer blocks (As the present study was performed using a preliminary small-scale screen, we found only one hit compound with a pyrimidine ring Table  that is Conceptualization, T.M. and S.U.; investigation, T.M., Y.O., S.M., J.Y., and S.U.; writing, T.M. and S.U.; funding acquisition, T.M. and S.U.; supervision, T.M. and S.U.The authors declare that there are no conflicts of interest.


Clade 2.3.4.4b highly pathogenic avian influenza (HPAI) A(H5N1) viruses that are responsible for devastating outbreaks in birds and mammals pose a potential threat to public health. Here, we evaluated their susceptibility to influenza antivirals. Of 1,015 sequences of HPAI A(H5N1) viruses collected in the United States during 2022, eight viruses (~0.8%) had a molecular marker of drug resistance to an FDA-approved antiviral: three adamantane-resistant (M2-V27A), four oseltamivir-resistant (NA-H275Y), and one baloxavir-resistant (PA-I38T). Additionally, 31 viruses contained mutations that may reduce susceptibility to inhibitors of neuraminidase (NA) (n = 20) or cap-dependent endonuclease (CEN) (n = 11). A panel of 22 representative viruses was tested phenotypically. Overall, clade 2.3.4.4b A(H5N1) viruses lacking recognized resistance mutations were susceptible to FDA-approved antivirals. Oseltamivir was least potent at inhibiting NA activity, while the investigational NA inhibitor AV5080 was most potent, including against NA mutants. A novel NA substitution T438N conferred 12-fold reduced inhibition by zanamivir, and in combination with the known marker N295S, synergistically affected susceptibility to all five NA inhibitors. In cell culture-based assays HINT and IRINA, the PA-I38T virus displayed 75- to 108-fold and 37- to 78-fold reduced susceptibility to CEN inhibitors, baloxavir and the investigational AV5116, respectively. Viruses with PA-I38M or PA-A37T showed 5- to 10-fold reduced susceptibilities. As HPAI A(H5N1) viruses continue to circulate and evolve, close monitoring of drug susceptibility is needed for risk assessment and to inform decisions regarding antiviral stockpiling. Highly pathogenic avian influenza (HPAI) A(H5N1) viruses persist in wild birds in many countries and cause deadly outbreaks in poultry and mammals worldwide. They are also known to cause sporadic human infections of various severity. Their circulation in wild birds is of public health concern as they may acquire the ability to effectively infect humans and transmit person-to-person . HPAI A. These vVaccines and antivirals are used to control seasonal influenza infections and are key components of pandemic preparedness. Antivirals can mitigate the impact of outbreaks in humans caused by zoonotic viruses for which vaccines may not be readily available. However, influenza viruses can become less susceptible to antivirals by acquiring genomic changes due to spontaneous mutations, gene reassortment, or selective pressure from antiviral treatment. To manage human influenza A virus infections, Food and Drug Administration (FDA) has approved antivirals from three classes: M2 blockers (adamantanes), neuraminidase (NA) inhibitors, and the cap-dependent endonuclease (CEN) inhibitor of the polymerase acid (PA) protein . Due to Evaluation of antiviral susceptibility is an integral part of the risk assessment conducted on influenza viruses that pose a threat to public health . In this50 (50% inhibitory concentration) of clade 2.3.4.4b viruses lacking any of the known or suspected NA markers of reduced inhibition  viruses, especially compared to oseltamivir (67-fold). Next, we evaluated the effect of each NA mutation on drug susceptibility by comparing the IC50 to the determined baseline. 50 of NA sequence-matched wildtype virus  viruses (n = 22) that were collected from different animal species and different genotypes , includi n = 15, . The medpe virus . This appe virus , whereaspe virus . Here, Npe virus . The twope virus , which ipe virus . Notablype virus . This ispe virus . By itsepe virus .50 (50% effective concentration) for viruses (n = 17) that lack flagged PA mutations  viruses seem to be slightly (1.2- to 5-fold) more susceptible to these antivirals than the reference human influenza A viruses. EC50s determined by HINT tended to be higher compared to those determined by IRINA, but this difference was small (~2-fold) (The A(H5N1) viruses were then tested for their susceptibility to the CEN inhibitors baloxavir (acid) and AV5116  using ceutations . The med~2-fold)  and S2. ~2-fold) . Expecte50s similar to that of the human A(H1N1)pdm09 virus with NA-H275Y. Not only did the dual mutation confer reduced inhibition by all NA inhibitors, but it also appeared to have lowered the NA activity of these viruses  for baloxavir susceptibility is arbitrary and more data are needed to improve laboratory data interpretation. A new cell culture-based assay, IRINA, provides improved throughput and its robustness was evident from the ability to test viruses displaying different growth kinetics. As clade 2.3.4.4b A(H5N1) viruses continue to circulate and evolve, close monitoring of their drug susceptibility is needed to inform clinical management in the event of human infections and to make informed decisions on the national stockpiling of antiviral medications.mmc1mmc2


Bacillus subtilis RecA, its negative (SsbA) and positive (RecO) mediators, and fork-processing (RadA/Sms). To understand how they work to promote fork remodeling, reconstituted branched replication intermediates were used. We show that RadA/Sms  binds to the 5′-tail of a reversed fork with longer nascent lagging-strand and unwinds it in the 5′→3′ direction, but RecA and its mediators limit unwinding. RadA/Sms cannot unwind a reversed fork with a longer nascent leading-strand, or a gapped stalled fork, but RecA interacts with and activates unwinding. Here, the molecular mechanism by which RadA/Sms, in concert with RecA, in a two-step reaction, unwinds the nascent lagging-strand of reversed or stalled forks is unveiled. First, RadA/Sms, as a mediator, contributes to SsbA displacement from the forks and nucleates RecA onto single-stranded DNA. Then, RecA, as a loader, interacts with and recruits RadA/Sms onto the nascent lagging strand of these DNA substrates to unwind them. Within this process, RecA limits RadA/Sms self-assembly to control fork processing, and RadA/Sms prevents RecA from provoking unnecessary recombination.Replication fork rescue requires  Replication fork progression is frequently impeded by physical barriers , shortage of DNA precursors, or collision with transcription elongation complexes or R-loops ,2,3,4. TEscherichia coli and Bacillus subtilis, which diverged >1.5 billion years ago and show a genetic distance larger than that between humans and plants, use similar pathways to overcome a replication stress, albeit with different hierarchal order.  In UV-irradiated E. coli cells, DNA synthesis drops within a few minutes, leading to a stable average number of replisomes ;,29;p < 0ed ssDNA  34];  when compared to the one observed by the RecA-cssDNA-SsbA complex (p < 0.01) when compared with RecA alone (When RecA (1 RecA monomer/12.5-nt), SsbA, and RadA/Sms were incubated with cssDNA, the maximum efficiency of ATP hydrolysis was increased by ~10-fold  its ATP hydrolysis is stimulated in the presence of cssDNA, and (ii) it fails to interact with RecA and to inhibit is ATPase activity [p < 0.01) (p < 0.01) when compared with the maximal rate of ATP hydrolysis by the RecA-cssDNA-SsbA complex  . The max complex . It is lPreviously, it has been shown that: (i) RecO cannot hydrolyze ATP , and (iiwt RadA/Sms or RadA/Sms C13A was not affected by the addition of a circa saturating RecO concentration (1 RecO monomer/100-nt)   . To avoiith RecA . These m < 0.01) . This inms K255R .p < 0.01) (p < 0.01) when compared with RecA-ssDNA-RadA/Sms complexes (RecO (1 RecO monomer/100-nt) was sufficient to partially displace SsbA (1 SsbA tetramer/66-nt) and significantly increased the maximal rate of ATP hydrolysis by RecA (1 RecA monomer/12.5-nt) . Howeveromplexes .From these data altogether we conclude that: (i) RadA/Sms-RecA, RadA/Sms K104R-RecA or RadA/Sms K255R-RecA complexes limit the ATPase of RecA and indirectly RecA dynamics; (ii) the presence of the two-component mediator (SsbA and RecO) cannot counteract RadA/Sms K104R or RadA/Sms K255R ; (iii) tp < 0.01) in the presence of ~6 and ~12 RadA/Sms, RadA/Sms C13A, or RadA/Sms K104A hexamers/cssDNA molecule, respectively (To further test whether RadA/Sms or its mutant variants (RadA/Sms C13A (unable to interact with RecA) or RadA/Sms K104A (unable to hydrolyze ATP)) can affect the enzymatic activity of RecA, a three-strand recombination assay was used. As revealed in ectively . Alternaectively . It is lwt cells, but it forms static foci in the ΔrecG context, where branched intermediates accumulate [RecA forms foci that colocalize with the stalled replisome in >90% of cells after UV-irradiation ,22. RadAcumulate ,40,41. Icumulate . To test65, SSB35, SSB15, and SSB7 binding modes) [The size site of RadA/Sms is unknown. To design the HJ-Lag substrate, we first analyzed the efficiency of unwinding of duplex DNAs with increasing 5′-tail lengths . RadA/Smg modes) ,48. RecAg modes) ,50,51. TRadA/Sms·ATP or RadA/Sms C13A·ATP (10 nM) bound and unwound the nascent lagging-strand of the HJ-Lag DNA substrate with similar efficiency A,B.wt RadA/Sms was replaced by RadA/Sms C13A  (wt RadA/Sms (or RadA/Sms C13A) once it is loaded on the 5′-tailed HJ-Lag DNA.To test the hypothesis, the combined action of RecA and SsbA over RadA/Sms (or RadA/Sms C13A) was analyzed by changing the order of protein addition B,C. Firsp < 0.01)  D, lane 5 < 0.01) . When RaFrom these data, it is likely that: (i) SsbA and RecA polymerizing toward the junction reduce RadA/Sms loading on the 5′-tail of HJ-Lag DNA, and additionally inhibit RadA/Sms unwinding of the nascent lagging-strand and the intermediates; and (ii) RecA and SsbA cannot outcompete a preformed RadA/Sms-HJ-Lag DNA complex.When a replication fork stalls due to a lesion on the lagging-strand, it may form a HJ-like structure with a nascent leading-strand longer than the nascent lagging-strand (HJ-Lead DNA) upon reversal . It has a to g) can be envisioned to explain how RadA/Sms in concert with RecA unwinds the nascent lagging-strand of HJ-Lead DNA (a) traces of a contaminating 3′→5′ ssDNA exonuclease in the RecA preparation may degrade the 3′tail of the HJ-Lead DNA to expose the 5′-tailed strand, with subsequent RadA/Sms self-loading; (b) dynamic RecA may partially open the duplex DNA at the junction to generate a RadA/Sms entry site (c) RadA/Sms may activate RecA to remodel the HJ substrate leading to flapped intermediates; (d) RecA bound to the 3′-tailed nascent leading-strand of HJ-Lead DNA might fray the nascent lagging-strand to promote RadA/Sms self-loading to the end of the nascent lagging-strand (e) in vivo RadA/Sms may be in a monomer–hexamer equilibrium in solution, and RecA may interact with and construct a ring-shaped RadA/Sms hexamer around the nascent lagging-strand from monomeric subunits (ring-maker strategy); (f) in the absence of ssDNA, RadA/Sms may adopt an open-spiral hexameric structure, as reported for LonAEco in the absence of a substrate [g) a hexameric RecA right-handed ring [Eco helicase or the β sliding clamp [a) to (f) and favored the ring-breaker hypothesis (condition g), unveiling a novel role of RecA. The sequence of the events to activate unwinding of the nascent lagging-strand—RecA complexed with hexameric RadA/Sms before interacting with the SsbA-ssDNA complex or RecA bound to ssDNA recruiting RadA/Sms on the nascent lagging-strand—is still unclear.Eight different scenarios (Lead DNA : (a) tratry site ; (c) Radg-strand ; (e) in ubstrate , and Recded ring  with a 1ng clamp ,55. As sIt has been shown that RecA·ATP neither nucleates nor polymerizes on the SsbA-ssDNA complexes ,42. Thenp < 0.01)  . A furth < 0.01) ; and (iip > 0.1)  ReIn a 40 min reaction, RadA/Sms bound to the 5′-tail of HJ-Lag DNA fully unwound the DNA substrate, but SsbA A, lane 9Then, we analyzed whether RecO and SsbA affect RecA activation of RadA/Sms-mediated unwinding of the HJ-Lead DNA B. RecO dIn previous sections, it was shown that: (i) RadA/Sms binds the 5′-tail of HJ-Lag DNA and unwinds the substrate, with RecA and its accessory proteins outcompeting RadA/Sms for binding to the DNA substrate ; and ii. DNA strRecA nucleated on a 15-nt long ssDNA does not hydrolyze ATP, enters into a “paused” state ,56, and wt RadA/Sms is replaced by RadA/Sms C13A, no unwinding was observed. It is likely that: (i) RecA nucleated toward the junction on the gapped forked-Lag DNA or away from the junction on the gapped forked-Lead DNA is necessary and sufficient to activate RadA/Sms to unwind the nascent lagging-strand of both DNA substrates, and (ii) RecA interacts with and modulates the conformation of hexameric RadA/Sms and loads it in cis or in trans on the nascent lagging-strand , RadA/Sms-mediated unzipping of the nascent lagging-strand was not observed  saturating SsbA bound to gapped ssDNA, perhaps in the SsbA15 binding mode, outcompetes RadA/Sms unwinding at a RadA/Sms:RecA:SsbA 1:5:15 molar ratio; (v) RadA/Sms, in concert with RecA, as a mediator, interacts with and facilitates RecA loading on the SsbA-ssDNA gapped DNA substrate; and (vi) about 6 RecA monomers in solution may open a hexameric ring and, upon interaction with 5 RecA nucleated at the 15-nt long gapped forked-Lag or forked-Lead DNA, assemble RadA/Sms on the nascent lagging-strand.From the data presented in B. subtilis respond to a replicative stress by forming RecA foci, in concert with SsbA and RecO, that colocalize with the replisome  at a stalled replication fork [wt cells but static foci in ΔrecG cells, a mutation that causes accumulation of branched intermediates [ion fork ,22. RadAmediates ,57. In tmediates ,45. Secomediates . In factmediates .From previous studies and the data presented here, six ideas can be drawn. First, cytological analyses have shown that SsbA, when travelling with the replisome, may load RecO, RecG, or PriA at a stalled replication fork ,18. RecOSecond, SsbA interacts with and loads RecO onto a lesion-containing gap, then RecO partially displaces SsbA and generates the substrate for RecA nucleation . Here, RThird, RecA neither nucleates nor polymerizes on the SsbA-ssDNA complexes ,29,30. Hcis or in trans onto the nascent lagging-strand to render a 3′-fork DNA specific for replication restart. This mechanism mimics loading of the replicative helicase DnaBEco by DnaCEco self-oligomerized as a right-handed helical filament by a ring-breaking strategy [Fourth, RecA·ATP (or RecA·ATPγS) protects the nascent strands from degradation and loads RadA/Sms on the nascent lagging-strand of stalled forks (or of reversed forks with a nascent leading-strand longer than the nascent lagging-strand) by a poorly understood mechanism A,B,D. Restrategy .Fifth, activated RadA/Sms unwinds the nascent lagging-strand of stalled forks A,B or ofFinally, RadA/Sms bound to the 5′ tail of HJ-Lag DNA unwinds the nascent lagging-strand to render a 3′-fork DNA substrate upon spontaneous remodeling . Here, RB. subtilis, different motor proteins process replicative stress intermediates [In conclusion, as reported in other systems, in mediates . HoweverE. coli BL21(DE3) (pLysS) cells bearing pCB1020 (radA), pCB1035 (radA C13A), pCB1038 (radA K104R), pCB1037 (radA K104A), pCB1040 (radA K255R), pCB722 (ssbA), or pCB669 (recO) encoding the respective gene under the control of a rifampicin-resistant promoter (PT7) were used to overproduce their respective proteins [B. subtilis BG214 cells bearing pBT61 (recA) were used to overproduce RecA [proteins ,59,60,61uce RecA .All chemicals used were analytical grade. Polyethyleneimine, IPTG , DTT, ATP, ATPγS, and dATP were from Merck . DNA ligase, DNA polymerase, and DNA restriction enzymes were from New England Biolabs . DEAE, Q-, and SP-Sepharose were from Cytiva . Hydroxyapatite was from Bio-Rad , phosphocellulose was from Whatman , and the Ni-column was from Qiagen .−1 cm−1, respectively, at 280 nm, as described [3COO)2, 50 μg/mL bovine serum albumin (BSA), and 5% glycerol).The protein RadA/Sms and its variants RadA/Sms C13A, RadA/Sms K104R, RadA/Sms K104A and RadA/Sms K255R (49.4 kDa), RecA (38.0 kDa), SsbA (18.7 kDa), and RecO (29.3 kDa) were expressed and purified as described ,42,47,63escribed . Protein−1 cm−1 at 260 nm for ssDNA and dsDNA, respectively, and are expressed as moles of nt or as moles of DNA molecules.The nucleotide (nt) sequence of the oligonucleotides used is indicated in the 5′→3′polarity: J3-1, CGCAAGCGACAGGAACCTCGAGAAGCTTCCGGTAGCAGCCTGAGCG GTGGTTGAATTCCTCGAGGTTCCTGTCGCTTGCG; J3-2-110, CGCAAGCGACAGGA ACCTCGAGGAATTCAACCACCGCTCAACTCAACTGCAGTCTAGACTCGAGGTTC CTGTCGCTTGCGAAGTCTTTCCGGCATCGATCGTAGCTATTT; J3-2-110-5, AAG TCT TTCCGGCATCGATCGTAGCTATTTCGCAAGCGACAGGAACCTCGAGGAATTCAA CCACCGCTCAACTCAACTGCAGTCTAGACTCGAGGTTCCTGTCGCTTGCG; J3-3, C GCAAGCGACAGGAACCTCGAGTCTAGACTGCAGTTGAGTCCTTGCTAGGACGGA TCCCTCGAGGTTCCTGTCGCTTGCG; J3-4, CGCAAGCGACAGGAACCTCGAGGGA TCCGTCCTAGCAAGGGGCTGCTACCGGAAGCTTCTCGAGGTTCCTGTCGCTTGC G; 170, AGACGCTGCCGAATTCTGGCTTGGATCTGATGCTGTCTAGAGGCCTCCAC TATGAAATCG; 171, CGATTTCATAGTGGAGGCCTCTAGACAGCA; 173, AGCTCAT AGATCGATAGTCTCTAGACAGCATCAGATCCAAGCCAGAATTCGGCAGCGTCT; 172, TGCTGTCTAGAGACTATCGATCTATGAGCT; 171-15, CGATTTCATAGTGGA; 172-15, ATCGATCTATGAGCT; 170-ER, AGACGCTGCCGAGGATCCCGGGTACCAAT CATAGCAATTGCAGCCTCCACTATGAAATCG; 173-ER, AGCTCATAGATCGATAG TGTATTAAGTGAGGATTGGTACCCGGGATCCTCGGCAGCGTCT; 171-ER, CGATTT CATAGTGGAGGCTGCAATTGCTAT; 172-ER, CTCACTTAATACACTATCGATCTAT GAGCT; 173-5, CAGCATCAGATCCAAGCCAGAATTCGGCAGCGTCT; 173-10, CTAG ACAGCATCAGATCCAAGCCAGAATTCGGCAGCGTCT; 173-15, AGTCTCTAGACAG CATCAGATCCAAGCCAGAATTCGGCAGCGTCT; 173-20, TCGATAGTCTTAGACAG CATCAGATCCAAGCCAGAATTCGGCAGCGTCT. In The ATP hydrolysis activity of RecA, RadA/Sms (or of its mutant variants), and PcrA was assayed in the presence of RecA mediators via an NAD/NADH coupled spectrophotometric enzymatic assay . Since R340 absorbance were converted to ADP concentrations and plotted as a function of time [wt RadA/Sms, or its mutant variant RadA/Sms C13A operates mostly under steady-state conditions, the maximal number of substrate-to-product conversion per unit of time (Vmax) was measured. t-tests were applied to analyze the statistical significance of the data. At longer times, ATP hydrolysis seems to decrease, but this is solved by increasing the concentration of the ATP regeneration system, suggesting that at very late times it is rate limiting.The rate of ATP hydrolysis was measured in buffer A containing 5 mM ATP and an ATP regeneration system  for 30 min at 37 °C . The ord of time . Acceptiw/v) PAGE. Gels were run and dried prior to phosphor-imaging analysis. The bands were quantified using ImageJ (NIH). t-tests were applied to analyze the statistical significance of the data.The different forked DNA substrates (0.5 nM) used were incubated with increasing concentrations of RadA/Sms, its mutant variant RadA C13A, or RecG in the presence of the indicated concentrations of RecA, RecO, or SsbA for 15 min at 30 °C in buffer A containing 2 mM ATP in a 20-μL volume. The reactions were deproteinized by phenol-chloroform; DNA substrates and products were precipitated by NaCl and ethanol addition and subsequently separated using 6% (KpnI-cleaved pGEM3 Zf(+) dsDNA (20 μM in nt) and the homologous circular 3199-nt ssDNA (10 μM in nt) were pre-incubated with the indicated protein or protein combination in buffer A containing 5 mM ATP or dATP (5 min at 37 °C). Then, a fixed RecA and a fixed or variable wt or mutant RadA/Sms concentration were added and the reaction was incubated for fixed or variable times at 37 °C. An ATP regeneration system (8 units/mL creatine phosphokinase and 8 mM phosphocreatine) was included in the recombination reaction. After incubation of the recombination reaction, samples were de-proteinized as described [lds) substrate and the appearance of joint molecule (jm) intermediates and nicked circular (nc) products were quantified from the gels using a Geldoc system and the Image Lab software , as described [jm and nc are shown (% of recombination).The 3199 bp escribed  and fracescribed . When in2+ matrix microcolumn, and the FT was collected. After extensive washing, the retained proteins in the Ni2+ matrix were eluted with 50 µL of buffer A containing 400 mM imidazole. The proteins were separated by 10% SDS-PAGE, and gels were stained with Coomassie Blue .In vitro protein–protein interactions were assayed using affinity chromatography as earlier described . His-tag


QCL.qau-4BS and QCL.qau-4DS were detected, which could explain 9.1%–22.2% of the phenotypic variances across environments on Rht-B1 and Rht-D1 loci, respectively. Several studies have reported that Rht-B1b may reduce the length of wheat CL but no study has been carried out at molecular level. In order to verify that the Rht-B1 gene is the functional gene for the 4B QTL, an overexpression line Rht-B1b-OE and a CRISPR/SpCas9 line Rht-B1b-KO were studied. The results showed that Rht-B1b overexpression could reduce the CL, while loss-of-function of Rht-B1b would increase the CL relative to that of the null transgenic plants (TNL). To dissect the underlying regulatory mechanism of Rht-B1b on CL, comparative RNA-Seq was conducted between Rht-B1b-OE and TNL. Transcriptome profiles revealed a few key pathways involving the function of Rht-B1b in coleoptile development, including phytohormones, circadian rhythm and starch and sucrose metabolism. Our findings may facilitate wheat breeding for longer coleoptiles to improve seedling early vigor for better penetration through the soil crust in arid regions.Wheat coleoptile is a sheath-like structure that helps to deliver the first leaf from embryo to the soil surface. Here, a RIL population consisting of 245 lines derived from Zhou 8425B × Chinese Spring cross was genotyped by the high-density Illumina iSelect 90K assay for coleoptile length (CL) QTL mapping. Three QTL for CL were mapped on chromosomes 2BL, 4BS and 4DS. Of them, two major QTL  Triticum aestivum L.) is one of the most important food crops in the world, providing large amounts of starch, rich protein and dietary fiber for humans  and Rht2 (Rht-D1b) combined with the increased application of chemical fertilizer greatly promoted the increase of wheat yield, which was called the “Green Revolution” of wheat  reduces the CL through multiple pathways such as phytohormones, circadian rhythm and starch and sucrose metabolism. These results provide valuable information for wheat breeding of longer coleoptiles to improve the seedling early vigor and penetration through soil crust in arid regions.Here, we demonstrated that 2:10 RILs derived from the cross of Zhou 8425B × Chinese Spring were used in this study. Zhou 8425B (Pedigree: Zhou 78A/Annong 7959) and Chinese Spring are an elite facultative wheat line and Chinese landrace, respectively. Zhou 8425B contains two dwarfing alleles Rht-B1b and Rht-D1b and has a short coleoptile length (CL) of about 3.3 cm. Chinese Spring contains two wildtype alleles Rht-B1a and Rht-D1a, which contribute to a long CL of about 4.8 cm. Seeds were sampled from plants grown and harvested at Shijiazhuang of Hebei Province and Qingdao of Shandong Province during the 2020–2021 and 2021–2022 cropping seasons, respectively. Good-quality seeds without any visible damage were selected for all lines. Seeds of all parental and progeny lines were sown in cylindrical pots (100 mm high and 80 mm in diameter) at a sowing depth of 2 cm below the soil surface. The CL was determined from the scutellum to the tip of the coleoptile.A total of 245 Fhttps://isbreeding.caas.cn/index.htm). Quantitative trait loci (QTLs) mapping was conducted using IciMapping 4.1 software with inclusive composite interval mapping (ICIM) algorithm  of Rht-B1b (GenBank: MG681100.1) was overexpressed in a hexaploid wheat cultivar Fielder (Rht-B1b and Rht-D1a) under driving by maize ubiquitin promoter (All primers were listed in Rht-B1b. The sgRNA (PAM-guide sequence 5’-GGAGCCGTTCATGCTGCAG-3’) was designed to target conserved regions of Rht-B1b. The resultant construct was transformed into immature embryos by the Agrobacterium tumefaciens (Rht-B1b overexpression lines (Rht-B1b-OE) and Rht-B1b CRISPR/SpCas9 edited lines (Rht-B1b-KO) were evenly sown in ten 10 cm (top diameter) × 8.9 cm (height) plant pots with a sowing depth of 2 cm below the soil surface. Before the coleoptile broke the ground, coleoptile tips and whole coleoptiles of ten TNL and Rht-B1b-OE plants were collected and immediately put into liquid nitrogen for RNA-Seq. Each group included three biological replicates.To study the association between efaciens . Sixty g® reagent, and mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. The first strand cDNA was synthesized using random hexamer primer and RNase H. Subsequently, the second strand cDNA synthesis was obtained using DNA Polymerase I and RNase H. Library preparation for RNA-Seq was conducted by Novogene and sequenced on an Illumina Novaseq platform with 1 ug of total RNA (http://www.novogene.com/).Total RNA of three biological replicates was extracted using the TRIzolP-value < 0.05 found by DESeq2 were assigned as differentially expressed genes (DEG). GO and KEGG enrichment analysis of DEGs were implemented by the TBtools  for coleoptile length (CL). Based on data averaged across all environments, CL ranged from 2.5 to 6.1 cm with an average of 4.0 cm. CL showed continuous variation in RIL population and had a high heritability of 0.86   were upregulated/downregulated by Rht-B1b in the transcriptome of whole coleoptile and coleoptile tips , which is gibberellin-insensitive protein in wheat  of the National Center for Biotechnology Information in the BioProject with accession number PRJNA936995.DX: Project administration, Investigation, Visualization, Writing, Editing and Funding acquisition. QH: Investigation, Data curation, Writing. TY: Investigation, Data curation. XL: Investigation, Data curation. HQ: Investigation. YW: Investigation. CJ: Investigation. WL: Investigation. XD: Investigation. JZ: Investigation. HZ: Investigation. ZH: Resources XX: Resources. SC: Review, Supervision and Resources. WM: Review, Supervision, and Funding acquisition. All authors contributed to the article and approved the submitted version.


Subacute sclerosing panencephalitis (SSPE) is a fatal neurodegenerative disease caused by measles virus (MV), which typically develops 7 to 10 years after acute measles. During the incubation period, MV establishes a persistent infection in the brain and accumulates mutations that generate neuropathogenic SSPE virus. The neuropathogenicity is closely associated with enhanced propagation mediated by cell-to-cell fusion in the brain, which is principally regulated by hyperfusogenic mutations of the viral F protein. The molecular mechanisms underlying establishment and maintenance of persistent infection are unclear because it is impractical to isolate viruses before the appearance of clinical signs. In this study, we found that the L and P proteins, components of viral RNA-dependent RNA polymerase (RdRp), of an SSPE virus Kobe-1 strain did not promote but rather attenuated viral neuropathogenicity. Viral RdRp activity corresponded to F protein expression; the suppression of RdRp activity in the Kobe-1 strain because of mutations in the L and P proteins led to restriction of the F protein level, thereby reducing cell-to-cell fusion mediated propagation in neuronal cells and decreasing neuropathogenicity. Therefore, the L and P proteins of Kobe-1 did not contribute to progression of SSPE. Three mutations in the L protein strongly suppressed RdRp activity. Recombinant MV harboring the three mutations limited viral spread in neuronal cells while preventing the release of infectious progeny particles; these changes could support persistent infection by enabling host immune escape and preventing host cell lysis. Therefore, the suppression of RdRp activity is necessary for the persistent infection of the parental MV on the way to transform into Kobe-1 SSPE virus. Because mutations in the genome of an SSPE virus reflect the process of SSPE development, mutation analysis will provide insight into the mechanisms underlying persistent infection. Subacute sclerosing panencephalitis (SSPE) is a rare but fatal disease in apparently healthy children, which develops 7 to 10 years after acute measles. Measles virus (MV) persistently infects the brain, accumulates mutations, and transforms into neuropathogenic SSPE virus. The World Health Organization is promoting vaccination against MV to achieve global eradication of measles; this is also the most effective approach for prevention of SSPE. The live MV vaccine strains do not cause SSPE and all SSPE viruses isolated thus far have been derived from wild-type MV strains, but it remains unclear why live MV vaccine strains do not cause SSPE. Recently hyperfusogenic mutations of the MV F protein were proved to account for the enhanced propagation of the virus in the brain leading to the neurovirulence. In this study we showed that suppression of viral RNA polymerase is implicated in the establishment of persistent wild-type MV infection within neurons. Further investigations of the mechanisms by which wild-type MV transforms into SSPE virus will shed light on the reason why MV vaccine strains do not acquire neurovirulence. Measles is a highly contagious acute infectious disease caused by measles virus (MV). Despite the availability of a safe and effective attenuated live virus vaccine, a worldwide resurgence of measles occurred between 2017 and 2019; the global incidence of measles reached 120 per million in 2019, and there were approximately 207,500 deaths . MV alsoMorbillivirus in the Paramyxoviridae family of the Mononegavirales order, contains a single-stranded, non-segmented, negative-sense RNA genome composed of six genes encoding eight proteins. The phospho (P) and large (L) proteins form the RNA-dependent RNA polymerase (RdRp) complex, which acts as a viral transcriptase or replicase by binding to the RNA genome encapsidated by the nucleocapsid (N) protein; the result is the RNase-resistant ribonucleoprotein (RNP) complex. The P gene encodes two non-structural proteins: V and C  on progeny virus production and growth in neuronal cells. As shown in Therefore, the suppression of RdRp activity restricted viral particle release and cell-to-cell spread among neuronal cells, suggesting that the L(triple) protein  promoted the establishment of persistent infection in the brain by limiting viral propagation.The L(triple) protein promoted persistent infection at the stage before onset of SSPE. To examine the effects of other mutations in the SSPEV-L protein on the RdRp activity of the L(triple) protein, we introduced mutations into the L(triple) protein . MutatioTo determine whether restoration of the RdRp activity of the L(triple) protein affects viral behavior, we generated rMVs harboring the L(triple) protein with the other mutations  based onBased on our findings, we constructed a model of the contributions of mutations in the SSPE virus Kobe-1 strain to the progression of SSPE . The finSSPE is a very rare late complication of MV infection that occurs in apparently healthy children, 7 to 10 years after acute measles –8. Althoet al.  . The plasmid p(+)MV323/SSPEV containing all genes of the Kobe-1 strain was constructed by replacing the SmaI–SalI fragment (nt 839–3364) of p(+)MV323/sMFHL with the corresponding region of the Kobe-1 strain.The plasmid p(+)MV323, which contained all MV ICB strain genes, was a gift from K. Takeuchi . The cDN016162)]  of p(+)MSalI fragment (nt 100–3364) or the SpeI–Eco47III fragment (nt 9176–15767) to replace the N–P genes unit or L gene unit of the MV ICB strain and the SSPE virus Kobe-1 strain, respectively, on the p(+)MV323-EGFP, p(+)MV323-EGFP/SSPEV, p(+)MV323-EGFP/sM, and p(+)MV323-EGFP/sMFH plasmids. To introduce the triple mutations  or triple plus additional mutations into the L protein of the rMVs, the SpeI–Eco47III fragment (nt 9176–15767) of the plasmids p(+)MV323-EGFP, p(+)MV323-EGFP/sM, or p(+)MV323-EGFP/sMFH was replaced with the corresponding fragment from pGEM/MV-L(triple) or pGEM/MV-L(triple) with the additional mutations described below.The plasmid p(+)MV323-EGFP, which contained all MV ICB strain genes and an additional EGFP transcriptional unit, was a gift from Y. Yanagi . The plaSpeI–Eco47III fragment of p(+)MV323-EGFP was cloned into the pBluescript II SK (-) vector to obtain pBS/MV-L. Next, the EcoRI–EagI fragment of pBS/MV-L was cloned into the pGEM vector to produce pGEM/MV-L for MV-L protein. pCA7/SSPEV-N, pCA7/SSPEV-P-ΔCV, and pGEM/SSPEV-L expressing the N, P, and L proteins of the SSPE virus Kobe-1 strain, respectively, were described previously [Renilla luciferase gene fragment into the pcDNA3 vector. Primer sequences used to construct these plasmids are available upon request.To construct pCA7/MV-N for expression of the N protein of MV, the N gene fragment (nt 108–1685) was amplified by PCR using p(+)MV323-EGFP and cloned into the pCA7 vector. pCA7/MV-P-ΔCV was constructed by cloning the P gene fragment (nt 1807–3330) of the MV ICB strain with nucleotide substitutions causing C and V protein deletion into the pCA7 vector. The eviously . To intrMonolayers of Vero/hSLAM cells in 24-well plates were infected with serially diluted virus samples. After incubation for 1 h at 37°C, the virus samples were removed, and medium containing 100 μg/mL fusion-inhibiting peptide  was added to block secondary infections. After 60 h, spots expressing EGFP were counted using a fluorescence microscope ; the number of the fluorescent spots was regarded as plaque-forming unit (PFU) .2 PFU of each recombinant chimeric virus in a 20 μL suspension of B95a cells. After inoculation, clinical signs were observed daily, and moribund mice were euthanized.BALB/c suckling mice, purchased from CLEA Japan, Inc. , were observed for health condition for a week and used prior to the age of 3 weeks. Mice were anesthetized, then intracerebrally inoculated with 7 × 10http://imagej.nih.gov/ij/) and expressed as cell-to-cell fusion activity.Vero/hSLAM cells cultured in T25 flasks were infected with cell-free rMVs and incubated at 37°C. At the indicated time points, photographs of representative syncytia were obtained using a fluorescence microscope. The percentage area occupied by syncytia in the images was quantified using ImageJ software (Monolayers of Vero/hSLAM cells in 24-well plates were infected with rMVs at a multiplicity of infection (MOI) of 0.04. After 2 h of incubation at 37°C, the virus samples were removed, and medium containing 100 μg/mL of fusion-inhibiting peptide was added. At 48 h post-infection, total RNA was extracted from virus-infected Vero/hSLAM cells using a NucleoSpin RNA kit (Macherey-Nagel); cDNA was prepared by reverse transcription using ReverTra Ace (Toyobo) in accordance with the manufacturer’s protocol, using specific primers for mRNA, (-) genomic RNA, and (+) genomic RNA. qPCR was performed using FastStart Essential DNA Green Master Mix and LightCycler 96  at 95°C for 600 s, followed by 45 cycles of 95°C for 10 s, 55°C for 20 s, and 72°C for 20 s. Melting curve analysis was performed after amplification. Relative expression levels of viral RNAs were calculated by the ΔΔCt method, using β-actin mRNA as the internal control. Primer sets for the target genes were described previously .g for 10 min at 4°C, and supernatants were collected. A small amount (24 μL) of each cell extract was mixed with sodium dodecyl sulfate (SDS) loading buffer. To detect the M protein, cell extracts were concentrated by chloroform/methanol precipitation using the method of Saito et al. [g for 5 min at 4°C. The protein pellet was washed with methanol, then mixed with SDS loading buffer. For collection of cell-surface samples, 800 μL of each cell extract were mixed with Streptavidin Sepharose High Performance  and incubated at 4°C for 2 h. The adsorbed biotinylated protein was used as the cell-surface sample.Vero/hSLAM cells were infected with rMVs as described above for qPCR. At 48 h post-infection, cells were incubated with 0.5 mg EZ-Link Sulfo-NHS-SS-Biotin  at room temperature for 30 min, then lysed in 1 mL of lysis buffer  at 4°C for 1 h. Next, lysates were centrifuged at 13,000 × o et al. . BrieflySamples were separated by SDS-polyacrylamide gel electrophoresis in 12% polyacrylamide gels, then electroblotted onto polyvinylidene difluoride membranes. Proteins were detected by incubating the membranes with a rabbit polyclonal antibody against MV-F protein , mouse mRenilla luciferase-expressing pcDNA3/R-Luc, then incubated at 37°C. At 24 h post-transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System  and Centro XS3 LB960  in accordance with the manufacturers’ protocols. Relative RdRp activity was calculated through division of firefly luciferase activity by Renilla luciferase activity. In Subconfluent monolayers of Vero/hSLAM cells in 24-well plates were transfected with 80 ng of the N protein-expressing pCA7/MV-N or pCA7/SSPEV-N; 120 ng of the P protein-expressing pCA7/MV-P-ΔCV, pCA7/SSPEV-P-ΔCV, or pCA7/MV-P-ΔCV with a mutation; 300 ng of the L protein-expressing pGEM/MV-L, pGEM/SSPEV-L, or pGEM/MV-L with mutations; and 350 ng of the MV mini-genome plasmid p18MGFLuc01 encoding the firefly luciferase gene (a gift from K. Komase) , along wg for 5 min at 4°C, the numbers of infectious cell-free viruses in supernatant were measured as described above for virus titration.Monolayers of Vero/hSLAM cells in 24-well plates were infected with cell-free rMVs at a MOI of 0.04. After 2 h of incubation at 37°C, virus samples were removed; the cells were washed with phosphate-buffered saline and incubated at 37°C. The culture medium was collected and replaced with fresh medium every 24 h. After the medium had been centrifuged at 2,000 × Comparisons between two groups were conducted using unpaired two-tailed Student’s t-tests. P-values < 0.05 were considered indicative of statistical significance.We performed the log-rank tests using the survival package in the R Software to analyze differences between survival curves. If a p-value was less than 0.05, the difference between the survival curves was considered statistically significant.S1 FigA) Enlargement of syncytia formed by rMV-infected Vero/hSLAM cells. Vero/hSLAM cells were infected with the EGFP-expressing cell-free rMVs in B) Spread of rMV infection in human neuronal cells. SH-SY5Y cells were infected with the EGFP-expressing cell-free rMVs in ((TIFF)Click here for additional data file.S2 FigA) Schematic of the genomes of the EGFP-expressing rMV/sMFH variants after exchanges of N, P, and L genes between the MV ICB strain and the SSPE virus Kobe-1 strain. (B) Viral cell-to-cell fusion. Vero/hSLAM cells were infected with the rMVs in (A). After incubation at 37°C for 36 h, a syncytium derived from a single infected cell was observed and photographed under a fluorescence microscope. Magnification, ×200. Cell-to-cell fusion was quantified as in C) Viral propagation in human neuronal cells. SH-SY5Y cells were infected with the rMVs in (A). After incubation at 37°C for 72 h, the spread of infection from a single infected cell was observed and photographed under a fluorescence microscope. Magnification, ×200. Spread of viral infection was determined as in ((TIFF)Click here for additional data file.S3 FigA) or the SSPEV-M protein (B) in Vero/hSLAM cells were infected with rMVs bearing the MV-M protein ((TIFF)Click here for additional data file.S4 FigA) Viral RNA levels in cells infected with rMVs harboring the L(triple) gene and its variants. Vero/hSLAM cells were infected with the rMVs in B) Surface F protein expression of cells infected with rMVs. Vero/hSLAM cells were infected with the rMVs in ((TIFF)Click here for additional data file.S5 FigA) Representative photographs of viral cell-to-cell fusion. Vero/hSLAM cells were infected with the rMVs in B) Representative photographs of viral propagation in neuronal cells. SH-SY5Y cells were infected with the rMVs in ((TIFF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Automatic whole brain and lesion segmentation at 7T presents challenges, primarily from bias fields, susceptibility artifacts including distortions, and registration errors. Here, we sought to use deep learning algorithms (D/L) to do both skull stripping and whole brain segmentation on multiple imaging contrasts generated in a single Magnetization Prepared 2 Rapid Acquisition Gradient Echoes (MP2RAGE) acquisition on participants clinically diagnosed with multiple sclerosis (MS), bypassing registration errors.Brain scans Segmentation from 3T and 7T scanners were analyzed with software packages such as FreeSurfer, Classification using Derivative-based Features (C-DEF), nnU-net, and a novel 3T-to-7T transfer learning method, Pseudo-Label Assisted nnU-Net (PLAn). 3T and 7T MRIs acquired within 9 months from 25 study participants with MS (Cohort 1) were used for training and optimizing. Eight MS patients (Cohort 2) scanned only at 7T, but with expert annotated lesion segmentation, was used to further validate the algorithm on a completely unseen dataset. Segmentation results were rated visually by experts in a blinded fashion and quantitatively using Dice Similarity Coefficient (DSC).Of the methods explored here, nnU-Net and PLAn produced the best tissue segmentation at 7T for all tissue classes. In both quantitative and qualitative analysis, PLAn significantly outperformed nnU-Net (and other methods) in lesion detection in both cohorts. PLAn's lesion DSC improved by 16% compared to nnU-Net.Limited availability of labeled data makes transfer learning an attractive option, and pre-training a nnUNet model using readily obtained 3T pseudo-labels was shown to boost lesion detection capabilities at 7T. Volumetric segmentation of brain magnetic resonance images (MRI) is now a commonly used post processing step enabling noninvasive quantitative analysis of disease progression in various neurological and neuropsychiatric diseases such as multiple sclerosis (MS), Alzheimer's disease, human immunodeficiency viral infection, and depression  methods, especially convolutional neural networks (CNNs) derived from the U-Net framework, have become state-of-the-art for brain MRI segmentation in recent years , entails pre-training an nnU-Net model with readily obtained pseudo-label data derived from scans at a lower field strength (3T), then fine-tuned with limited 7T expert-drawn labels (transfer learning) to optimize lesion segmentation performance. The performance of this method was compared against commonly available methods on 3T and 7T data and manually drawn lesion masks in patients clinically diagnosed with MS.The Natural history of MS study protocol was approved by the institutional review board of our institute (NCT00001248), and all participants provided written informed consent.In this retrospective study, 33 MS patients were included in two cohorts to train and evaluate nnU-NET, PLAn, and C-DEF-7T. Cohort 1 consists of 25 patients . Inclusion criteria for Cohort 1 was available 3T and 7T scans within 9 months of each other; no clinical disease progression or new lesions between their 3T and 7T scans . 3T and 7T scans from this cohort will be referred to as Cohort 1-3T and Cohort 1-7T. Cohort 2 included 8 MS patients  scanned only at 7T , and was mainly used to validate the segmentation technique.1 maps .3T images, acquired on a Skyra system with a 32-channel head coil, included 3D T1-weighted (T1w) images , 3D FLAIR , and 2D PD/T2 . 7T images were acquired on a Magnetom system equipped with a 1-channel transmit/32-channel receive coil  and Tn = 5) and a testing group (n = 20). Segmentation labels for training/validation were created for cohort 1-3T using C-DEF as previously described  as described elsewhere  as well for comparison (C-DEF 7T). The only changes made to the original pipeline were that the percentile filter bias correction  were obtained by down-sampling 7T T1w MP2RAGE scans to 1.0 mmn = 5) utilizing the publicly available nnU-Net package . The only significant modification made to nnU-Net 7T was to disable largest-connected-component post-processing, which was not helpful for this task.An nnU-Net segmentation model (nnU-Net 7T) was trained using cohort 1-7T's training/validation group (n = 5). Fine-tuning was conducted for 125 epochs  as preliminary analysis found it to be the shortest training regime that remained stable. The initial learning rate was 1 × 10 – 4 (default = 1 × 10 – 2), which was determined by cross-validation to be the best compromise between preserving the pre-trained weights and learning new information from the 7T data. No other settings were altered, and no model layers were frozen during fine-tuning. The fine-tuned model was then applied to the 7T MP2RAGE scans of the remaining unseen participants to obtain cross-validation results.Finally, a 3T to 7T transfer learning model was trained for 7T segmentation (PLAn 7T). The C-DEF 3T model segmentation labels (referred as pseudo-labels for this purpose) and 3T MP2RAGE scans were used to pre-train an nnU-Net model using the 3D full-resolution U-Net configuration and default settings. Transfer learning was achieved by loading the pre-trained model weights and preprocessing settings , then fine-tuning the network with the manually edited 7T labels and 7T MP2RAGE scans from cohort 1-3T training group  rated the quality of segmentation for each tissue class and skull stripping for each participant scan. A subjective rating scale from 1 to 5 was used, with the best segmentation being ranked as a 5 . The meafslstats utility from FSL , and all data reported are in the form of mean ± standard deviation.Statistical analysis was performed using PRISM version 9.3.1. After checking the normality assumption using the Shapiro-Wilk test, we employed repeated-measures ANOVA using Dunnett's method to compare mean metric differences of segmentation methods to either the 3T reference or PLAn (as appropriate), corrected for multiple comparisons. A corrected First, skull stripping performance was evaluated using qualitative ratings and Bland-Altman plots, which compared total intracranial volumes (TIV) to the reference AFNI 3dSkullStrip performed on 3T MP2RAGE (AFNI 3T). The nnU-Net 7T and PLAn 7T methods produced excellent skull stripping results throughout the brain –D. BlandWhile AFNI 7T and AFNI 3T produced similar skull stripping boundaries in some brain regions, such as dorsal regions , other rr = 0.92, CI: , p < 0.001, slope = 1.2). This is consistent with prior observations that C-DEF 3T under-strips the skull in dorsal regions, leading to slightly lower CSF volumes than expected. FreeSurfer 7T produced degraded temporal lobe and cerebellum segmentation, mislabeling sulcal gray matter (GM) as CSF throughout the ventral regions . C-DEF 7T had much better CSF segmentations due to good cortical boundaries (All volumetric comparisons are in reference to the 3T segmentation outputs. The D/L methods (nnU-Net 7T and PLAn 7T) significantly outperformed both C-DEF 7T and FreeSurfer 7T for all tissue segmentation classes from cohort 1 . They su regions , D. Thisundaries , B, althundaries . The nnUundaries . As a reundaries .r = 0.95 CI: , p < 0.001, slope = 0.68). C-DEF 7T, on the other hand, failed to detect most lesions throughout the brain . Meanwhile, FreeSurfer 7T demonstrated significant lesion sensitivity, with a moderately strong lesion volume correlation of r = 0.90 , both of which were significant increases compared to C-DEF 7T  from 0.68 for nnU-Net 7T to 0.90 for PLAn 7T and corresponded to an improved expert lesion rating of 3.92  for PLAn 7T, compared to 3.74  for nnU-Net 7T (a 5% increase).Despite its substantial advantages over other 7T methods, nnU-Net 7T still produced noticeable deficiencies in lesion detection , C. To ap < 0.0001) when compared to C-DEF 3T. In addition, PLAn showed stronger slope which was closer to unity  significantly improved lesion segmentation performance by leveraging the readily available 3T pseudo-labels to mitigate limited 7T label availability. Importantly, PLAn-7T only requires a 3T cohort for training, but not in application of the segmentation.For all segmentation tasks at 7T, D/L recon methods clearly outperformed C-DEF and FreeSurfer. nnU-Net's excellent skull stripping is consistent with prior studies which established the efficacy of U-Net-derived CNNs for brain extraction in general  and FreeSurfer (detailed GM segmentation). Nevertheless, FreeSurfer's cortical segmentations in the temporal lobe and cerebellum suffered from degradation. Given this degradation's localized and systematic nature, it is likely due to inadequate mitigation of the strong 7T bias fields, as previously found in FreeSurfer analyses at ultra-high fields  without large-scale manual annotation, which can be incredibly expensive and tedious. These labels cannot be treated as a perfect 7T gold standard due to the inherent differences between segmentations at 3T and 7T. Additionally, a FLAIR contrast was included at 3T but not 7T. Previous research has shown that lesion segmentation volumes derived from FLAIR contrasts are higher than from TPLAn was pre-trained on pseudo-labels derived from cohort 1-3T and then initially evaluated on the same participants scanned at 7T (cohort 1-7T). Then, we compared the lesion segmentation performance of PLAn and nnU-Net on an unseen cohort (cohort 2) with reference to manually drawn labels. Future work will apply PLAn on multi-site data, including other disease cohorts, and refining the pseudo-label and gold standard generation method.n = 20), which may indicate that utilizing more pseudo-labeled data for pre-training could also boost performance.One area for further investigation is whether a larger training data set (source or target domain) would significantly improve the segmentation of lesions or other classes. A recent study of transfer learning for subcortical segmentation found that while just a few (1 to 3) images were sufficient for fine-tuning in most cases, smaller structures such as the amygdala and accumbens were most improved by additional data for fine-tuning (Kushibar et al., In this study, we developed and applied a state-of-the-art deep learning method to MP2RAGE 7T MR images to obtain fast and reliable whole-brain segmentations. By employing only multi-contrast techniques such as MP2RAGE, it is readily scalable and adaptable to use in various conditions and performs robustly against any registration errors. The performance of 7T lesion segmentation was boosted, and training label scarcity for lesion segmentation was overcome by incorporating a pre-training step using results from a robust 3T segmentation as pseudo-labels before fine-tuning a transfer-learning method. We present these findings as a blueprint for acquiring fast, accurate, and valuable volumetric markers from 7T MRI data for use in clinical and research settings.® Xeon® CPUs with 256 GB RAM running CentOS 7.7. All nnU-Net and PLAn models were implemented on a computing cluster with NVIDIAÒ v100-SXM2 GPUs, each with 32 GM VRAM and 24 Intel® Xeon® CPUs, each with 64 GB RAM.All C-DEF and FreeSurfer processing was implemented on a computing cluster with 64x Intelhttps://github.com/hdieckhaus/PLAn-7T.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. PLAn software is available at The studies involving humans were approved by Institutional Review Board of the National Institutes of Health. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


NL-G-F MAPTP301S mouse that at 6 months of age exhibits robust Aβ plaque accumulation, intense MAPT pathology, strong inflammation and extensive neurodegeneration. The presence of Aβ pathology potentiated the other major pathologies, including MAPT pathology, inflammation and neurodegeneration. However, MAPT pathology neither changed levels of amyloid precursor protein nor potentiated Aβ accumulation. The APPNL-G-F/MAPTP301S mouse model also showed strong accumulation of N6-methyladenosine (m6A), which was recently shown to be elevated in the AD brain. M6A primarily accumulated in neuronal soma, but also co-localized with a subset of astrocytes and microglia. The accumulation of m6A corresponded with increases in METTL3 and decreases in ALKBH5, which are enzymes that add or remove m6A from mRNA, respectively. Thus, the APPNL-G-F/MAPTP301S mouse recapitulates many features of AD pathology beginning at 6 months of aging.The study for the pathophysiology study of Alzheimer’s disease (AD) has been hampered by lack animal models that recapitulate the major AD pathologies, including extracellular β-amyloid (Aβ) deposition, intracellular aggregation of microtubule associated protein tau (MAPT), inflammation and neurodegeneration. We now report on a double transgenic APP The hallmark pathologies of Alzheimer’s disease (AD) consist of the accumulation of neuritic plaques composed of β-amyloid (Aβ), the accumulation of neurofibrillary tangles (NFTs) composed of microtubule associated protein tau , inflammation and neurodegeneration . MutatioModels relying only on genetic modification of amyloid precursor protein (APP) develop abundant Aβ plaques but produce little MAPT pathology beyond modest increases in phosphorylation . Mouse mThe absence of robust MAPT pathology in mouse models expressing only endogenous MAPT likely derives from the low aggregation propensity of murine MAPT , 6. ThisMultiple groups have explored crossing transgenic APP mouse over-expression models with tau mouse models –19. The NL-G-F KI and the PS19 P301S MAPT mouse lines were crossed. The resulting mouse model, termed APPNL-G-F/MAPTP301S, develops many aspects of AD pathology. The APPNL-G-F/MAPTP301S mouse exhibits a progressive increase in Aβ load, neuritic plaques, all major forms of tau pathology, as well as exhibiting enhanced microglial activation, astrogliosis, neurodegeneration and a progressive loss in cognitive function. This model also exhibits other key elements of AD pathology including inflammation and elevated levels of N6-methyl-adenosine (m6A) tagged RNA, which has recently been shown to change strongly with disease progression . Th. ThNL-G-a of CA3 . The celNL-G-F/MAPTP301S model. Inflammatory cells are readily evident in the area around Aβ plaques (NL-G-F or APPNL-G-F/MAPTP301S model. These results are consistent with published work indicating that Aβ pathology elicits a phenotype termed “Disease Associated Microglia” while MAPT pathology elicits microglial responses exhibiting weaker cytokine production [Increasing evidence points to a key role for inflammation in AD. Many AD-linked genes appear to enhance disease risk by affecting the biology of microglia. For instance, TREM2 is one of the strongest risk factors for AD, and evidence suggests that TREM2 acts to recognize Aβ pathology and direct microglial responses toward the pathology , 55. The plaques . These moduction .6A parallel those reported by our group previously [6A correlated with levels of MAPT pathology. This finding is consistent with the observation that MAPT pathology co-localizes with the RNA binding protein, HNRNPA2B1, which functions as an indirect m6A reader. The results presented above in 6A levels in the context of MAPT pathology, Aβ pathology and combined MAPT and Aβ pathology. The results are clear in that m6A levels follow MAPT pathology. A previous manuscript examining m6A in an AD model utilizing over-expression of only APP, which is a model that exhibits Aβ accumulation without corresponding MAPT pathology. The data presented above suggest that the absence of MAPT pathology in this model produced a corresponding absence of m6A accumulation [6A in disease. The increases in m6A observed correlate with MAPT pathology are show a corresponding association with increased levels of METTL3 and reduced levels of ALKBH5, which are the enzymes that respectively add and remove m6A from mRNA. More writing combined with reduced erasure leads to increased levels of m6A.The increases observed for meviously . We obsemulation . Our stu6A were most evident in neurons, the results in 6A in the APPNL-G-F/MAPTP301S model. Such results are consistent with emerging studies showing that m6A regulates inflammation, macrophages and also astrocytosis [6A modulation might also be applied towards regulation of inflammation in AD; indeed a recent study observed that conditional knockout of METTL3 in microglia attenuated inflammation and Aβ accumulation in a mouse model based on Aβ injection [Although changes in mocytosis –61. Suchnjection .NL-G-F/MAPTP301S model described in this manuscript takes advantage of the APPNL-G-F KI line, but utilizes the human MAPTP301S line in order to achieve robust MAPT pathology. Our results show that this model provides an appealing alternative to the double APPNL-G-F x MAPT KI model, which develop both MAPT pathologies very slowly [NL-G-F/MAPTP301S model thus provides a useful compromise. This model exhibits strong Aβ accumulation and strong inflammation, while avoiding artifacts associated with APP or presenilin over-expression. The APPNL-G-F/MAPTP301S model also possesses the benefits arising from strong expression of human MAPT with the resulting rapid development of robust MAPT pathology, strong m6A accumulation and, importantly, significant neurodegeneration.The field is increasingly moving towards use of KI models. The APPy slowly . The cur


Changes are indicated with underlined text as follows:Fig. S3 C), we failed to detect a prominent proliferation of Tregs in the contactless set-up.Despite the ability of C1-B in secreting IL10 .To better delineate the C1-B population, we immunophenotyped LN-derived cells from BQ mice 30 d after COL2 immunization (Fig. S1 F, from three different cohorts were employed.Serum samples, used in These changes do not alter the conclusions of the manuscript. The errors appear in PDFs downloaded before September 20, 2023.


Ablating phosphorylation at T4102 attenuates DNA-PKcs kinase activity and this destabilizes the interaction between DNA-PKcs and the Ku-DNA complex, resulting in decreased assembly and stabilization of the NHEJ machinery at DSBs. Phosphorylation at T4102 promotes NHEJ, radioresistance, and increases genomic stability following DSB induction. Collectively, these findings establish a key role for ATM in NHEJ-dependent repair of DSBs through positive regulation of DNA-PKcs.Ataxia-telangiectasia mutated (ATM) drives the DNA damage response via modulation of multiple signal transduction and DNA repair pathways. Previously, ATM activity was implicated in promoting the non-homologous end joining (NHEJ) pathway to repair a subset of DNA double-stranded breaks (DSBs), but how ATM performs this function is still unclear. In this study, we identified that ATM phosphorylates the DNA-dependent protein kinase catalytic subunit (DNA-PK  Complex to DSBs . DNA-PKcactivity . Once accs promotes DSB repair via the non-homologous end joining (NHEJ) pathway , DNA Ligase 4 (LIG4), and XRCC4-like factor (XLF) collectively stabilize at the DNA damage site via a number of protein-protein interactions and are modulated by DNA-PKcs kinase activity , is the kinase primarily responsible for signal transduction and cell cycle regulation in response to DSBs . Cells dn events . ATM plan events . Finallyn events .cs. The data show that DNA-PKcs is phosphorylated by ATM in its FATC domain at threonine 4102 in response to DSBs. Ablating phosphorylation at T4102 decreased DNA-PKcs kinase activity and results in destabilization of the interaction between DNA-PKcs and the Ku-DNA complex. Phosphorylation of DNA-PKcs at threonine 4102 results in stabilization of the NHEJ machinery at DSBs, leading to increased NHEJ efficiency and genomic instability. Our results establish a model in which phosphorylation of the FATC domain of DNA-PKcs by ATM promotes efficient NHEJ.Here, we uncover a novel function for ATM in NHEJ via phosphorylating DNA-PKcs wild-type, T4102A, and T4102D were cultured in Hyclone α-minimum Eagle's medium supplemented with 5% newborn calf serum, 5% fetal bovine serum (Corning), and 1X penicillin/streptomycin (Gibco). U2OS cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1× penicillin/streptomycin (Gibco). All cells were grown in an atmosphere of 5% CO2 at 37°C. To inhibit DNA-PKcs, ATM or ATR, cells were incubated for 2 h prior to irradiation with 5 μM NU7441 (SelleckChem), 5 μM KU55933 (SelleckChem) or 5 μM VE821 (SelleckChem), respectively. Enrichment of U2OS cells at G1 phase and S/G2 were performed via a double thymidine block as previously described (Chinese Hamster Ovary (CHO) V3 cells and V3 cells stably expressing DNA-PKescribed .137Cs irradiator (J.L. Shepherd and Associates) with a dose of 10 Gy, unless otherwise indicated in the figure.Cells were irradiated with γ-rays generated by a Mark 1 3]QVKCLMC. The phospho-specific antibodies were first passed through the corresponding unphosphorylated peptide-conjugated Sepharose CL-4B column (Pierce) to deplete IgGs that were not phospho-specific. The flow through IgGs were then affinity-purified using a phospho-peptide column. Eluted anti-pT4102 polyclonal antibodies were verified with phospho and non-phospho peptides via dot plot analysis.Anti-pT4102 polyclonal antibodies were generated by immunizing New Zealand white rabbits with KLH (keyhole limpet hemacyanin)-conjugated phosphopeptide GLSEET[POcs with alanine or aspartic acid using the pCMV-F2-K and pCMV-YFP-K as a template to generate mutated FLAG-tagged and YFP-tagged DNA-PKcs, respectively. The primers used were: T4102A: T4102A Forward 5′- TGGGCTTTCAGAAGAGGCTCAAGTGAAGTGCCT-3′ and T4102A Reverse 5′-AGGCACTTCACTTGAGCCTCTTCTGAAAGCCCA-3′ and T4102D: T4102D Forward 5′-GTGGGCTTTCAGAAGAGGATCAAGTGAAGTGCCTG-3′ and T4102D Reverse 5′-CAGGCACTTCACTTGATCCTCTTCTGAAAGCCCAC-3′.Site-directed mutagenesis was performed using PCR to substitute T4102 on DNA-PKcs phospho-T4102 (made in this study as described above), anti-DNA-PKcs phospho-S2056 , anti-ATM phospho-S1981 , anti-CHK2 phospho-T68 , anti-LIG4 , anti-Ku80 , anti-Ku70 , anti-XRCC4 , anti-XLF , anti-GFP , anti-KAP1 (Bethyl Laboratories A300-274A), anti-KAP1 phospho-S824 , anti-tubulin , anti-FLAG M2 , anti-phospho-H2AX (S139) , and anti-Histone H3 antibody . Mouse monoclonal antibodies against DNA-PKcs (Clone # 25-4) were produced in house. Secondary antibodies used include anti-mouse IgG (HRP-linked)  and anti-rabbit IgG (HRP-linked) . Image J (Version 1.53e) was used to quantify the intensities of protein bands in the immunoblots.Immunoblotting was performed as previously described . The folcs at T4102, V3 cells stably expressing YFP-tagged wild-type DNA-PKcs were irradiated with a dose of 10 Gy and allowed to recover for 30 min. The cells were then washed twice with cold PBS, harvested, and lysed using IP lysis buffer ). The lysates were sonicated on ice and then cleared of cellular debris by centrifuging at 20 000 × g for 30 min. 2 mg of total protein was incubated with the DNA-PKcs monoclonal antibody (25-4) and Protein A/G beads (Thermo Fisher) overnight at 4°C with mixing. The following day the beads were washed three times with lysis buffer and then twice with washing buffer . Following the final wash, the beads were divided two parts, with one resuspended in 1× SDS sample buffer and other treated with lambda protein phosphatase (λPP) (New England Biosciences) at 30°C for 30 min to remove phosphorylation events, the samples were washed, and finally resuspended in 1× SDS sample buffer. The samples were resolved via SDS-PAGE and phosphorylation at T4102 was assessed via immunoblotting.To detect phosphorylation of DNA-PK2, 0.4% NP-40, 0.6% Triton X-100, 1× protease inhibitor cocktail, 1× phosphatase inhibitor cocktail 2, 20 U/ml Benzonase (Novagen)) on ice. The lysates were sonicated on ice and then cleared of cellular debris by centrifuging at 20 000 × g for 30 min. 2 mg of total protein was incubated with 2 μg anti-GFP antibody, and 30 μl of Protein A/G magnetic agarose beads (Thermo Fisher) overnight with spinning at 4°C. The beads were washed with IP washing buffer  for 5 times, and boiled in 1× SDS sample buffer. The samples were resolved via SDS-PAGE and immunoblotting was performed for the proteins indicated in the figures.To investigate protein-protein interactions, CHO V3 cells and V3 cells complemented with WT, T4102A, or T4102D were irradiated with a dose of 10 Gy and allowed to recover for 10 min. Subsequently, the irradiated cells were washed three times in cold PBS, harvested, and lysed using IP Lysis buffer  and 30 μl Protein A/G beads for DNA-PKcs. After an overnight incubation at 4°C, the beads were washed twice with Purification Lysis Buffer, twice with Wash Buffer 1  and finally twice with Wash Buffer 2 . For ATM protein, half of beads were resuspended in 1× SDS sample buffer and the sample was resolved via SDS-PAGE, and the other half was for the in vitro phosphorylation assay. To remove phosphorylations associated with the purified DNA-PKcs protein, the beads were further equilibrated in 1× λPP reaction buffer (New England Biosciences) and treated with λPP at room temperature for 30 min. The sample was washed using the protocol described above. One-third of the beads containing DNA-PKcs was resolved via SDS-PAGE, and the rest were utilized for the ATM-mediated in vitro phosphorylation assay. The SDS-PAGE gel that resolved purified YFP-DNA-PKcs and FLAG-ATM was Coomaissie Blue stained to show purification of each protein.Purification of ATM from HT1080 cells stably expressing FLAG-tagged ATM and DNA-PKications . To actiIn vitro phosphorylation of DNA-PKcs by ATM was conducted by mixing beads containing YFP-DNA-PKcs with beads containing FLAG-tagged ATM or control beads. The bead mixtures were placed in Kinase Reaction Buffer  and incubated at 37°C for 1 h with mixing. The reactions were terminated by adding 1× SDS sample buffer and boiling the samples at 95°C for 5 min. The phosphorylation of DNA-PKcs at T4102 was then assessed by immunoblotting with the pT4102 antibody.cs kinase activity was assessed by examining phosphorylation of H2AX peptide in vitro, using a modified version of our published protocol , 0.1 μg sonicated herring DNA, 20 nM Ku70/80, 0.1 μg biotin-labeled H2AX (biotin-AVGKKASQASQEY) and 2.5 μg of nuclear extract from V3, V3 + DNA-PKcs WT, V3 + DNA-PKcs T4102A, or DNA-PKcs T4102D cell lines. After a 30 min incubation at 30°C, the reactions were terminated by the addition of 1 μl 0.5 M EDTA, and the biotinylated-H2AX peptide was captured using a SAM2 Biotin Capture Membrane (Promega) and the membrane was washed following the manufacturer's suggested protocol. H2AX phosphorylation was detected using an anti-H2AX pS139 antibody via immunoblotting and signal was quantified using ImageJ (1.53e). Background phosphorylation of H2AX was observed in the DNA-PKcs null V3 cell line and this was subtracted from the other samples’ readouts. 100% kinase activity was normalized using the V3 cells expressing WT human DNA-PKcs. The reported results are derived from three independent experiments.DNA-PKprotocol . BrieflyIR-induced 53BP1 foci kinetics were monitored in G1 cells as previously described with modifications ,26. Briecs WT, T4102A, T4102D, Ku80, XRCC4, XLF and PNKP in response to DSB induction was examined following laser micro-irradiation with a Carl Zeiss Axiovert 200M microscope with a Plan-Apochromat 63×/NA 1.40 oil immersion objective (Carl Zeiss) as previously described  and incubated with 10 μM BrdU. 24 h later, the medium was replaced with CO2-independent medium and placed in a chamber on the microscope that was set at 37°C. To generate laser-induced DSBs, a 365-nm pulsed nitrogen laser  was set at 80% of maximum power output and micro-irradiation was performed using the pulsed nitrogen laser. Time-lapse images were taken using an AxioCam HRm camera (Carl Zeiss). Carl Zeiss Axiovision software (v4.91) was used to measure fluorescence intensities of the micro-irradiated and control areas, and the resulting intensity of irradiated area was normalized to non-irradiated control area to obtain the alteration of the interested proteins as described previously  for 40 min at 4°C. The cells were then pelleted by centrifugation at 5000 × g and the resulting pellets were lysed with buffer 1 containing 50 mM HEPES pH 7.5, 50 mM NaCl, 0.05% SDS, 2 mM MgCl2, 10% glycerol, 0.1% Triton X-100, 1× protease inhibitor and phosphatase inhibitor cocktails, and 10 Units of RNase-free DNase I. The chromatin proteins were included in the supernatant after centrifugation at 18 000 × g. The protein concentration of each sample was measured using a Pierce BCA Protein Assay kit (Thermo Fisher). 20 μg of each fraction was resolved via SDS-PAGE, and then transferred to a PVDF membrane for immunoblotting.The accumulation of DNA damage response proteins to chromatin following IR-induced DNA damage was examined as previously described with some modifications . BrieflyCHO cells were seeded in triplicate at a density of 40 000 cells per well in 12-well plates. The following day, cells were transfected with reporter plasmids using Lipofectamine 3000 (ThermoFisher) according to the manufacturer protocol. For each cell line, one well was transfected with undamaged plasmid cocktail, one well was transfected with NHEJ plasmid cocktail, and one well was left untransfected. After 24 h, cells were trypsinized and analyzed by flow cytometry using an Attune NxT flow cytometer. Compensation and gating were established by running untransfected cells along with cells transfected with individual undamaged plasmids expressing wild type fluorescent proteins (single color controls) as described previously . Each exet al. and below:Reporter plasmid cocktails were as follows: undamaged plasmid cocktail: 50 ng pMax_BFP, 50 ng pMax_mOrange, 500 ng non-fluorescent carrier plasmid pDDCMV. NHEJ plasmid cocktail: 50 ng BFP_NHEJ, 50 ng pMax_mOrange, 500 ng non-fluorescent carrier plasmid pDDCMV. Reporter fluorescence was used as a measurement of NHEJ efficiency as described in Piett cs WT, T4102A or T4102D were mock treated or irradiated at doses of 1, 2, 4 or 6 Gy, or incubated with different concentration of etoposide or Camptothecin as indicated in the figure legend, and then plated on 60-mm plastic Petri dishes. After 8 days, cells were fixed with 100% ethanol and stained with 0.1% crystal violet in a 100% ethanol solution. Colonies were scored and the mean value for triplicate culture dishes was determined. Cell survival was normalized to plating efficiency of untreated controls for each cell type.Cell survival curves were obtained by measuring the colony-forming abilities of irradiated cell populations as previously described . CHO V3 To investigate genome stability following irradiation in CHO V3 cells and V3 cells complemented with WT, T4102A or T4102D, the cells were irradiated with a dose of 2 Gy and then allowed to recover in normal cell culture conditions for 24 hrs. Next, the cells were incubated with Colcemid at concentration of 0.1 μg/ml for 4 h and then harvested by trypsinzation. After washing with PBS at room temperature, the cells were then incubated with warmed (37°C) 75 mM KCl. Samples were processed and chromosomal abnormalities were scored as previously described .cs is composed of HEAT (Huntington-elongation factor 3A- PP2A subunit-TOR) repeats, which are differentiated into a unique N-terminal domain  and a central unit called the M-HEAT domain (amino acids 893-2801) and a C-terminal region that contains the kinase domain (amino acids 3565-4100), which is flanked N-terminally by the FAT  domain and C-terminally by the FATC  domain   domain . The FATno acids . Early wDNA-PKcs . This dr(pT4102) . Howevers Figure , but this Figure  34). An. Ancs ist Figure  7,35). ,35. cs in Figure . Conformcs is phosphorylated at this amino acid after DNA damage in cells, a phospho-specific antibody to T4102 was generated and validated using dot blot analysis (cs is phosphorylated at T4102 (pT4012) following exposure to ionizing radiation (IR) and the signal was lost when the sample was treated with lambda phosphatase, supporting that the antibody recognizes a phosphorylation event  cell line V3 with YFP-tagged wild-type DNA-PKcs (WT) or DNA-PKcs in which the phosphorylation site at T4102 was ablated via alanine substitution (T4102A) (cs at T4102 was observed after treatment with the topoisomerase 1 inhibitor camptothecin (CPT), but limited to no phosphorylation occurred following treatment with the DNA cross-linking agent mitomycin (MMC) and DNA alkylating agent methyl methanesulfonate (MMS)  . The sig(T4102A) . Further(T4102A) , with ob(T4102A) . This ph(T4102A) , and IR-(T4102A) . In addi(T4102A) . Modest ) Figure . As IR, e Figure . We next1 Figure . This dawith ATM . IR-induM Figure . Finallye of ATP . ATM auty Figure . Collectcs kinase activity. We complemented V3 cells with YFP-tagged WT, T4102A, and the DNA-PKcs mutation in which T4102 was mutated to aspartic acid to mimic phosphorylation at this amino acid (T4102D)  post-IR treatment that DNA-PKcs phosphorylates H2AX at S139 and KAP1 at S824 , but significantly reduced accumulation/retention of DNA-PKcs at the DNA damage site  . Subsequubstrate . We obse at S824 . Therefos Figure . Furthers Figure . This daD Figure . ConsistA damage  38–40)..cs kinas . Subseqs Figure . As ATM age site . Collectcs has been implicated in promoting the long-range synaptic complex and T4102 promotes stabilization of the DNA-PKcs-Ku complex, we next assessed if phosphorylation at this site affects the dynamics of the NHEJ machinery at DSBs. First, the recruitment of the core NHEJ factors Ku80, XRCC4 and XLF to laser-generated DSBs was examined in V3 cells complemented with FLAG-tagged DNA-PKcs WT, T4102A and T4102D  results in almost complete loss of NHEJ, which was rescued by expression of WT DNA-PKcs  and S2023, S2029, S2041, S2051 and S2056 (termed the S2056 or PQR cluster) and phosphorylation of both clusters is required for NHEJ and modulation of DSB repair pathway choice. The S2056/PQR and T2609/ABCDE phosphorylation clusters have opposing roles in protecting DSBs, with S2056/PQR protecting the DNA ends and T2609/ABCDE promoting DNA end processing (cs to the DNA-Ku complex, triggering its release from DSBs to allow access of DNA ends for processing, including the terminal ligation step of NHEJ (cs is modulated by phosphorylation at T3950 and S56/S72, whereas phosphorylation at T946/S1004 inhibits NHEJ without affecting the enzymatic activity of DNA-PKcs (cs phosphorylation in modulating its functionality, as we show that in response to DSBs, ATM phosphorylates DNA-PKcs in the FATC domain of the protein at T4102. This phosphorylation event promotes stabilization of the DNA-PKcs-Ku interaction and the stabilization of the NHEJ machinery at DSBs, both of which stimulate NHEJ.DNA-PK to DSBs . The bes of NHEJ . The kinDNA-PKcs . Here, wcs and ATM, and that they may cooperatively initiate DSB repair signaling and regulate DSB repair (cs and ATM leads to synthetic lethality in mice (cs and ATM phosphorylate many common targets required for DDR signaling and DSB repair, including H2AX, KAP1 and components of the NHEJ pathway, such as XLF and Artemis (cs at S3205 and at the T2609/ABCDE cluster in response to DNA damage (cs to allow DNA end ligation (cs also phosphorylates the T2609/ABCDE cluster in trans (cs may be context dependent. Moreover, ATM phosphorylation of DNA-PKcs overcomes DNA-PKcs-Ku inhibition of resection in vitro (cs and ATM and supports another positive role in ATM promoting NHEJ via phosphorylation of DNA-PKcs.A body of evidence shows that crosstalk occurs between DNA-PKB repair ,32. Firs in mice ,44. SecoA damage ,45. ATM-ligation . DNA-PKcin trans ,47,49,50in vitro . Lastly,in vitro . This stcs in its FATC domain at T4102 promotes stabilization of DNA-PKcs at DSBs, which supports assembly and maintenance of the NHEJ core complex at the DNA damage site. We found that ablating the T4102 site attenuates overall DNA-PKcs kinase activity and that this correlates with decreased interaction with the Ku-DNA complex and stabilization of the NHEJ machinery. We hypothesize that the decrease in the NHEJ core complex stability at the DNA damage site is due to the attenuated DNA-PKcs kinase activity in the T4102A mutant. This is supported by previous data showing that the transition from the NHEJ long-range to the short-range complex and recruitment of factors required for DNA end processing are modulated by DNA-PKcs kinase activity (cs induces long-range conformational changes in DNA-PKcs that are important for mediating DNA-PKcs-mediated protein-protein interactions with the core NHEJ factors. The structural predictions suggest that conformational changes within DNA-PKcs will be required to accommodate phosphorylated T4102, which may be facilitated by residues from the FAT, kinase, or FATC sub-domains in this region. The modeling does not provide evidence that phosphorylation at T4102 will directly affect the interaction between DNA-PKcs with the Ku heterodimer or another core NHEJ factor, but we hypothesize that this phosphorylation event may induce an allosteric conformational change that allows the stabilization of the long-range complex. Furthermore, ablating the T4102 phosphorylation site attenuates the recruitment of the DNA end processing factor PNKP to DSBs, suggesting that DNA-PKcs activity and/or a conformational change upon DNA-PKcs phosphorylation at T4102 may promote the processing of unligatable DSBs. We postulate that this allows ATM protein to ensure the complete repair of DSBs by stabilizing the NHEJ complexes to favor the processing and correct joining of DNA ends.A significant number of single molecule studies have produced data showing the makeup of the NHEJ machinery at DNA ends. In particular, two synaptic complexes, the long-range and short-range complexes, have been identified and these complexes are believed to protect the DSB ends and then position them for ligation, respectively ,7. Althoactivity ,30. It igkad505_Supplemental_FilesClick here for additional data file.