There is a great fundamental interest in charge dynamics of PbS quantum dots, as they are promising for application in photovoltaics and other optoelectronic devices. The ultrafast charge transport is intriguing, offering insight into the mechanism of electron tunneling processes within the material. In this study, we investigated the charge transfer times of PbS quantum dots of different sizes and non-quantized PbS reference materials by comparing the propensity of localized or delocalized decays of sulfur 1s core hole states excited by X-rays. We show that charge transfer times in PbS quantum dots decrease with excitation energy and are similar at high excitation energy for quantum dots and non-quantized PbS. However, at low excitation energies a distinct difference in charge transfer time is observed with the fastest charge transfer in non-quantized PbS and the slowest in the smallest quantum dots. Our observations can be explained by iodide ligands on the quantum dots creating a barrier for charge transfer, which reduces the probability of interparticle transfer at low excitation energies. The probability of intraparticle charge transfer is limited by the density of available states which we describe according to a wave function in a quantum well model. The stronger quantum confinement effect in smaller PbS quantum dots is manifested as longer charge transfer times relative to the larger quantum dots at low excitation energies. By measuring the resonant S-KLL Auger decay we investigate the attosecond charge transfer in PbS quantum dots of different sizes. The results show both intra- and interparticle charge transfer, where the former shows a strong QD size dependence. This size-bandgap relation is a consequence of quantum confinement, first described in the 1980s by the group of L. Brus.6,7 For solar cells of the third generation, this has proven to be an exceptional quality since using different nanocrystal sizes and thus different bandgaps, the material may be tuned to absorb a specific range of electromagnetic spectrum.Quantum dots (QD) are a well-known type of semiconductor material often used in fields where precise control of the light emitted or absorbed by them is necessary. The most common applications include LEDs, biosensors, detectors, transistors, imaging and photovoltaic devices such as solar cells.8,9 and the stability in air is reported to be over 300 h.10 Still, greater improvements are necessary to reach industrial standards. In attempt to contribute to the improvements, many studies are done on the charge generation, charge transport from QDs to the electrodes of the cell and charge dynamics in general, using time-resolved pump-probe methods.11–18 In these methods, the excitation of the particle usually happens from the valence bands, which are delocalized all over the particle. Consequently, it is difficult to determine which atoms are being excited, and if they are in any way affected by the presence of the created vacancies. In the present study, we investigated X-ray core-excited states that decay through auto-ionization. In contrast with the common pump-probe methods, when using core-excitations from deep core levels there is very little to no overlap with the valence band, and the influence of the present vacancies is diminished.One of the currently most studied quantum dot solar cell (QDSC) material are halide capped lead sulfide (PbS) quantum dots with roughly 1.3 eV bandgap and devices using these QDs have reached power conversion efficiency of above 13%,2,3L2,3 Auger electron energy range reveal the dynamics of this excited in resonant Auger spectroscopy (RAS). Finally, by applying RAS in core hole clock spectroscopy (CHCS) using the lifetime of the core hole (created by the resonant excitation) as an internal clock19 it is possible determine the charge transfer (CT) times in the quantum dot PbS and the continuous PbS film samples. This initial charge transfer is localized and unaffected by holes in the valence band. Herein, we present the impact that the quantum dot size i.e. quantum confinement has on the ultrafast (sub-femtosecond) charge transfer dynamics in PbS materials. Few studies have been performed using CHCS to study photovoltaic materials20–23 and our study concerns PbS quantum dots in comparison to PbS in thin films.As a first step, we determined the resonant region of PbS material by exciting the S K absorption edge (to S p states) which allowed us to study the unoccupied states in the PbS samples using X-ray absorption spectroscopy (XAS). In this excitation energy range, around the S K edge, mapping of the S KL2 and PbBr2, which were ordered from TCI.All chemicals in the synthesis were purchased from Sigma-Aldrich except for the PbI24 with several modifications depending on the nanocrystal size, with main parameters summarized in −1.The PbS colloidal QDs are synthesized as previously reported24 while the natural galena crystal was ordered from Crystal Cave Rocks shop and cleaved in air just before being stored into vacuum chamber.The PbS reference thin film was prepared as previously reported,Details on the sample preparation and spin-coating of quantum dot films can be found in the ESI.†25 at the KMC-1 beamline26 at the BESSY II electron storage ring operated by the Helmholtz-Zentrum Berlin für Materialien und Energie, where the available photon energies range from 2 keV to 12 keV. The photon energy was selected using a Si double crystal monochromator and the X-rays were focused on the sample using a parabolic glass capillary. The pressure in the analysis chamber was low 10−8 mbar and a VG Scienta R4000 hemispherical electron energy analyzer was used to measure the kinetic energies of the electrons emitted from the sample. The binding energy scale was calibrated by measuring the Au 4f7/2 core level of a gold foil mounted on the manipulator and setting its position to 84.0 eV binding energy, while the photon energy scale for the XAS data was calibrated using the first and third order X-rays from the monochromator and measuring Au 4f photoelectron spectra. The XAS data was collected using a Bruker XFlash 4010 fluorescence detector mounted on the same experiment chamber. All of the measurements were always carried out on fresh sample spots.Hard X-ray Photoelectron Spectroscopy (HAXPES), resonant Auger spectroscopy (RAS) and X-ray absorption (XAS) measurements were carried out at the HIKE endstation24 quantum dot samples of three different sizes, with a PbI2 surface treatment, and a cleaved natural galena PbS crystal , the sample architecture is presented in The samples used in this experiment are continuous PbS thin film (PbS ref) prepared as previously reported,17,27 and has been assigned to existence of electron states within the bandgap, which could be surface trap states or defects.28,29 In the absorption spectra the QDs showed clear differences in exciton peak positions 1.79 eV, 1.29 eV and 0.81 eV center energy, as obtained from a least squares curve fitting with a Gaussian distribution  using a photon energy of 3000 eV. All the core levels from PbS QD samples are shifted to higher BE in relation to the core levels from PbS reference sample , which m32 In fact, thinking of these ions as spheres and calculating the surface/volume ratio in terms of simplest geometry, the surface/volume ratio for PbS 2 nm, PbS 3 nm and PbS 5 nm becomes 3, 2 and 1.2 nm−1, respectively, while the volume ratio of the surface layer consisting of iodide ions compared to the PbS volume in quantum dot is 0.8, 0.5 and 0.3. Calculation details may be found in the ESI.24). As shown in Fig. S4,24 The S 1s of the natural galena also shows a clean surface with small additional contributions to the S 1s spectrum  states (due to lower population of states corresponding to the S 3p states) are not necessarily found at the same position as the fitted onset of the VB spectra.34 From the photoelectron spectroscopy measurements of quantum dots of various sizes and application of density of states (DOS) model, the authors propose the following correction for the valence band maximum position:Eg is the bandgap of the quantum dot. The VBM calculated using this correction gives VB values which reflect the size difference more clearly  spectra, which selectively probes the S 3p orbitals. In order to be plotted together with the VB spectra , right, eference , which c2,3L2,3 Auger signal in resonant conditions at photon energies ranging from 2460 to 2500 eV. Core hole clock spectroscopy (CHCS)19 is an application of Resonant Auger spectroscopy (RAS) for determining electron charge transfer times on the femto- to attosecond (10−15 to 10−18 s) timescale. This is achieved by using the lifetime of the core hole (created by the resonant excitation of an electron) as an internal clock.19After mapping out the X-ray absorption upon excitation from S 1s unoccupied states, we investigated the S KL38 or sometimes localized and delocalized decay,39 respectively. In spectator decay, the electron in the conduction band stays localized during the Auger decay and the emitted electron has a constant binding energy , this is also referred to as the Raman channel. In the other process, charge transfer occurs before the decay of the core hole yielding the same final state configuration as a normal Auger decay would (however with a photon energy below the ionization threshold) meaning that the emitted electron thereby has a constant kinetic energy. Scanning the Auger region over a range of photon energies around the resonance, we obtain a resonant Auger map. In this Auger map the charge transfer (incoherent) channels are seen as constant kinetic energy features  therefore give the relative probabilities of the two processes (core hole decay without charge transfer and charge transfer followed by core hole decay), and their ratio is referred to as Raman ratio . One can consider these two processes as independent single exponential decays, where the decay time for the Raman channel is the core hole lifetime (τL) and the decay time for the normal Auger channel is the charge transfer time (τCT). The Raman ratio is then inversely proportional to the ratio of the decay times, and one can calculate the charge transfer (CT) times in our system according to:19,40The experiment begins with determining the absorption edge energy, in our case with XAS of the S K-edge. Around this photon energy is the region of interest for the resonant Auger study. Once an electron has been excited to the CB creating an excited state consisting of a core hole and an excited electron in the CB , the sys41 In the case of our experiments, we would be able to determine charge transfer times in the range of tens of attoseconds to tens of femtoseconds.The determination of charge transfer times therefore depends on how accurately the Raman ratio can be determined and on the core hole lifetime, which is 1.27 fs for S 1s.The RAS map of the PbS ref sample recorded over the S K-edge resonance in the kinetic energy region of the S KLL Auger is shown in the 2,3L2,3 Auger line (1D2 transition)42 along with two less intense channels ,42 marked with vertical lines, and focus in this paper is set on the most intense (main) Auger line. As described above, it is necessary to know the Raman ratio, which is given by intensity ratio of Raman and Auger feature from individual spectra of the resonant map, to determine the CT time. Therefore, the components of the spectra were manually deduced for each map, using the least squares curve fitting procedure described in the ESI.43–45 In order to get a meaningful fit, the Raman contribution was assigned two Voigt peaks with controlled widths (E) between the first Raman feature (at lower KE) and the 1D2 Auger peak  from the excited QD to an adjacent QD, whereas with intraparticle CT we are referring to the electron movement or delocalization within the same QD. The CT time in interparticle CT region (B) is similar for all samples but appears a bit faster in smaller QDs. This suggests that the electrons hop between small QDs easier with higher excess energy, i.e. at higher excitation energy. Contrastingly, in part A, the electrons are given excitation energy just above resonance and in this part, there is a clear difference in charge transfer time between the different PbS samples.In the map in d widths , left. Ad widths  were ploger peak . For calime data  we may d2 – reduced graphene oxide interface, explained by the formation of Schottky barrier at the interface.43 However as our QD films are roughly 250 nm thickness, and the probing depth at these photon energies should not be more than 13 nm in the sample, we disregard influence of the substrate/PbS interface. Thus, one may conclude that the constant CT time region in E). This can be explained with a quantum mechanics model,46 where in potential wells with finite barrier height , the number of allowed energy states a wave function can have is dependent on the width of the potential well, i.e. quantum dot size. For instance, if a wave function (an electron) is given enough energy to be transmitted from one potential well to another one (from one dot to another) and on its way it encounters a potential barrier (a ligand layer) there are two possible outcomes: the electron wave function tunnels through the barrier (interparticle transfer), or is reflected from the barrier into the well (intraparticle CT), as illustrated in Furthermore, a flat part of the CT time curve can be observed in region A with the CT time being constant for about 1 eV. This is most clearly seen for the 3 and 5 nm samples and also present, but not as clear, in the PbS ref sample. A similar CT time trend has previously been observed in a MoSE) within this region, which indicates that CT is less limited at these energies. For the PbS ref thin film the charge transfer time does decrease with increasing energy, which indicates an increase in available states with increasing energy. This could be due to band bending towards the surface caused by the iodide treatment, which makes the surface more n-type compared to the surface of the galena crystal , which implies a faster CT time at higher excitation energies. Moreover, comparing the polycrystalline PbS film and the galena crystal we note that whereas the latter has a constant tunneling probability in region of lower excitation energies above resonance, the polycrystalline film has a sharp increase in tunneling probability (CT).To summarize, we measured the ultrafast charge transfer times of quantum dot PbS, continuous PbS film and a natural crystal PbS material using Resonant Auger and core hole clock spectroscopy. Our results show that charge transfer in PbS QDs is slower at low excitation energies just above the resonance. In this region, charge transfer correlates with the diameter of the QDs and can be assigned to intraparticle CT. This can be qualitatively explained by smaller quantum dots having access to fewer states within the dot if considering the quantum dots as separated finite quantum wells with certain widths. However, if the excitation energy is above a certain threshold , the charge transfer tends toward unity with increasing energy, There are no conflicts to declare.RA-012-D2RA06091D-s001


The aim of this paper was to analyze the impact of the addition of different amounts of zirconium  on selected properties of AlSi5Cu2Mg aluminum alloy. This is a new alloy for cylinder head castings and has only been used for a relatively short time. The specificity of this alloy is its chemical composition—specifically the low permitted Ti content, which makes it impossible to refine the grain structure of this alloy using standard Al-Ti-B grain refiners. The aim of our ongoing research is to find a suitable alloying element that would positively mainly affect the mechanical and also physical properties of this alloy, which are crucial for complex automotive castings such as cylinder heads. The results of our research showed that increasing zirconium content had no effect on the increase in ultimate tensile strength, yield strength and hardness of as-cast samples. After T7 heat treatment, a more significant increase in UTS, YS and thermal conductivity occurred due to the precipitation of Cu- and Mg-rich strengthening precipitates. Zirconium-rich intermetallic phases were observed in the shape of separate thick needles or as a cluster of two crossed thinner needles. SEM observations showed that these phases crystallized near to the intermetallic phases based on Cu and Fe. Increasing the Zr content was accompanied by an increase in liquidus temperature, the density index and the area fraction of porosity values. In recent years, we have witnessed the automotive industry undergoing significant changes. Due to increasingly stringent regulations on emission standards, car manufacturers are forced to come up with technological solutions to reduce emissions and improve fuel efficiency. In addition to emission-improving solutions such as particulate filters, low-viscosity oils or fuels with bio additives, automakers are introducing to the market vehicles with alternative powertrains . The introduction of emission-reducing technological solutions also affects manufacturers of vehicle components . The above components are preferably made of aluminum alloy castings. This fact affects the research centers that have to develop new aluminum alloys that will meet the criteria for advanced automotive castings. In this way, the competitiveness and sustainability of production in the automotive sector is ensured ,2,3.Even though the current trend in the automotive industry is based on the promotion of electric cars, the world is far from ready for the full transition to electromobility. The high prices of electric cars, insufficient infrastructure of charging stations, lack of rare metals for battery production and the difficulties in recycling them are the factors for which fossil fuels and conventional combustion engines will not be completely displaced in the coming years .Conventional internal combustion diesel/petrol engines have undergone significant changes in recent decades. The common denominator of these changes is the so-called “downsizing”. This term expresses the effort of manufacturers to provide more efficient vehicles with lower emissions by reducing engine displacement and/or number of cylinders while maintaining or even enhancing engine performance. As a result, the specific output of an engine (describing the efficiency of an engine in terms of power-to-displacement ratio) is constantly increasing . This le2Si or Al2Cu) occurs, resulting in a decrease in the mechanical properties. Cylinder heads of modern engines are exposed to temperatures exceeding 200 °C and therefore it is necessary to focus on development of new alloys for such kind of use [Cylinder heads are among the most thermally stressed engine components. During operation, they must be able to withstand temperature changes, mechanical loads and the corrosive effects of exhaust gases. Commercially available cast aluminum alloys for cylinder heads (mostly based on the Al-Si-Cu-Mg system) can operate at temperatures up to 200 °C. Above this temperature, coarsening and dissolving of main strengthening phases . Of all transition metals, zirconium (Zr) has been the subject of various studies in recent years. The grain-refining effect of Zr and the thermal stability of Zr-containing intermetallic phases are attributes that predetermine its use in the development of new high-strength aluminum alloys operating at elevated temperatures above 200 °C ,12.Grain refinement of cast aluminum alloys is a basic metallurgical operation. The grain refinement mechanism is based on the introduction of inoculant particles in the form of master alloys, resulting in the nucleation initiation of α (Al) phase on the surface of these particles at small undercooling. Alloys with a finer grain structure may have improved mechanical properties, higher homogeneity and reduced possibility for hot tearing .0 is the solute content in the alloy and k0 is the partition coefficient.To express the effectiveness of a chemical element used as a grain refiner, a growth-restriction factor Q has been introduced. A higher value of the Q factor means that the grain refiner more effectively restricts grain growth and affects the formation of a fine-grained equiaxed structure. The growth-restriction factor can be determined as ,16,17,180 [s and cl represent the equilibrium solute contents of the solid and liquid at the growing interface.The Q factor value (and thus the efficiency of the grain refiner) is closely related to the segregation properties of the alloying element. Segregation properties can be described by the partition coefficient k0 , defined0 > 1, the solute is preferentially absorbed by the growing solid rather than being segregated ahead. In areas enriched by an alloying element, heterogeneous nucleation can take place. Therefore, a potential grain-refining element in aluminum alloys should have a partition coefficient greater than 1. Such elements include (in addition to the commonly used Ti) Zr, Mo and V ;Capability of forming strengthening intermetallic phases with trialuminide type AlLow solid solubility in Al, to maximize the equilibrium volume fraction of the dispersed phase, retard volume diffusion-controlled coarsening and prevent dissolution of precipitated phases;Low diffusivity in Al, to retard volume diffusion-controlled coarsening through which the precipitates are allowed to act as an effective barrier to dislocation movement at elevated temperatures.According to Knipling , aluminu3Zr phase with a cubic L12 structure. The peritectic Al-Zr system is characterized by a limited concentration of Zr in the solid solution after conventional solidification [Zirconium as an alloying element meets all three criteria. Zr-containing aluminum alloys gained popularity due to the formation of the trialuminide Alfication ,24,25.Slow diffusion of the alloying element in aluminum is key to maintaining the mechanical properties or retarding their decrease at elevated temperatures. Chemical elements from the group of transition metals  are characterized by very low diffusion in Al; therefore, aluminum alloys alloyed with such elements are particularly suitable for the production of alloys operating at elevated temperatures. The values of diffusivity D and activation energy Q are important parameters for assessing the thermal stability of precipitates containing a transition metal. As shown in The aim of our research was to investigate the effect of varying Zr addition on selected properties of the non-standardized AlSi5Cu2Mg alloy. This alloy was developed as a result of Korean automobile concern over the production of gasoline engine cylinder head castings. As this alloy has been used in production for a relatively short time, there is minimal (if not any) available research data about this alloy. This non-standardized AlSi5Cu2Mg alloy has been designed with a very specific chemical composition—low permitted Ti content, which practically makes it impossible to grain refine the microstructure via the standard AlTi5B1 grain refiner. The aim of our research was to find suitable alloying elements that would positively mainly affect the microstructure and mechanical properties of this alloy. Zr is experiencing increasing popularity as an alloying element due to its grain-refining effect and the possibility of increasing strength characteristics of aluminum alloys. This was our motivation to address the effect of Zr on selected properties of the AlSi5Cu2Mg alloy in our research. Most studies published so far have been focused on evaluating the impact of Zr on the mechanical properties and microstructure of standardized hypoeutectic Al alloys with 7 to 10 wt. % Si. The presented research was focused on the evaluation of Zr impact in the non-standardized AlSi5Cu2Mg aluminum alloy with low Si content (5 to 6.5 wt. %). Our research deals with a more complex evaluation of the non-standardized AlSi5Cu2Mg alloy. In addition to the influence of Zr on mechanical properties and microstructure, we also dealt with the influence of Zr on thermal conductivity, melt gasification or porosity formation. Based on this, we believe our research differs from others in the greater complexity of assessing the impact of Zr on selected properties of the hypoeutectic AlSi5Cu2Mg alloy..5 or AlSi10Mg(Cu), have been used and contain from 6 to 10 wt. % Si.Low Si content, in particular the lower limit of the Si range . So far,Limited Ti content (max. 0.03 wt. %). This limitation practically restricts the use of commercially available grain refiners based on Al-Ti-B. For a sufficient grain-refining effect of hypoeutectic Al-Si alloys, it is necessary to add 0.04 to 0.1 wt. % Ti.The hypoeutectic aluminum alloy AlSi5Cu2Mg in a pre-modified state  were produced by the introduction of Zr in the form of the AlZr20 master alloy. Chemical composition of the experimental alloys is given in The production of experimental alloys consisted of melting the primary alloy AlSi5Cu2Mg in an electric-resistant furnace. At 780 ± 5 °C, Zr was added in the form of the AlZr20 master alloy. The casting temperature was 745 ± 5 °C. Experimental samples were produced by gravity casting into a permanent metal mold with temperature ranging from 180 to 200 °C. Ten experimental samples were produced for each experimental alloy. Half of the samples were used for subsequent evaluation of selected properties in the as-cast state and half were heat treated by age hardening. Experimental samples were age hardened using T7 over-aging heat treatment consisting of solutioning at 500 ± 5 °C for 6.5 h, followed by quenching into hot water (80–90 °C). Artificial aging was carried out at 250 ± 5 °C for 4 h, followed by air cooling. The T7 mode is often used for heat treatment of castings with complex geometry (such as cylinder heads) to achieve a relatively stable microstructure and mechanical behavior during operation. T7-type heat treated aluminum alloys are characterized by scarification of some degree of strength to improve other characteristics. Strength characteristic are mainly “traded off” to improve dimensional stability of cylinder heads (to secure trouble-free operation at elevated temperatures) and to lower residual stresses ,30.Mechanical properties of the experimental alloys were evaluated based on the tensile strength test according to ISO 6892-1 standard using an Inspekt desk 50 kN testing machine. For each experimental alloy, 10 tensile test bars were produced (five for the as-cast state and five for T7 heat treatment), as shown in 2SO4 etchant.Microstructural evaluation was performed on samples with the best combination of mechanical properties. The microstructure was observed using a NeoPhot 32 optical microscope and TESCAN LMU II scanning electron microscope with a BRUKER EDX analyzer. Samples for metallographic evaluation were prepared by coarse and fine wet grinding, two-step polishing using a 3 and 1 µm diamond paste and subsequent etching with HThe area fraction of porosity and the length of Zr-rich intermetallic phases were evaluated using the graphic software Quick Photo Industrial 3.1. Five random places on each experimental sample were chosen to evaluate these properties.Thermal conductivity of the experimental alloys was evaluated by the alloy manufacturer’s method. This method was based on the measurement of the electrical conductivity using a Sigma Check 2 conductometer with a touch sensor. Five measurements were performed for each experimental alloy. Obtained values of electrical conductivity (σ) were used in the empirical Formula (3) to calculate the values of thermal conductivity (λ).The solidification path of the experimental alloys was evaluated by thermal analysis. Equipment for the thermal analysis consisted of a K-type thermocouple located in the middle of the cylindrical shaped mold. Using a LabView 2 Hz software, measured values  during solidification were recorded. A cooling curve and its first derivative were generated from the obtained data.Hardness of the experimental alloys was evaluated by Brinell method using a Brinell Innovatest Nexus 3000 hardness tester with 5 mm diameter ball with a load of 250 kp (2451.6 N) and a dwell time of 10 s. A total of 5 measurements were performed for each experimental alloy.Characteristic solidification temperatures depending on the addition of different amounts of Zr obtained from the cooling curves and their first derivatives are shown in liq) increased  can adversely affect the hot-tearing susceptibility of the experimental alloys.With increasing Zr addition, liquidus temperature . Density index evaluation belongs to the basic procedures to evaluate the quality of the melt. Based on the DI value, it is possible to determine the degree of hydrogen gasification of the melt. DI determination was based on a density comparison of two samples, one of which solidified at atmospheric pressure and the other in vacuum (80 mbar for 4 min). It is clear from the measured values  that theThe effect of Zr on porosity was also evaluated by determining the area fraction of porosity . On each experimental alloy, five different locations were selected for the measurement. The pores  after the addition of 0.05 and 0.10 wt. % Zr by 6%, and after the addition of 0.15 wt. % Zr by 4%. The probable cause was crystallization of hard Al3Zr phases, which contributed to the overall increase in hardness. After heat treatment, ductility values decreased from 2 to 1% regardless of Zr addition.Mechanical properties of the experimental alloys with varying Zr addition in the as-cast state and after T7 heat treatment are shown in When developing new types of aluminum alloys for complex castings in the automotive industry, great emphasis is placed not only on mechanical properties but also on sufficient thermal conductivity. This is especially important in the cylinder head castings of modern turbocharged combustion engines with direct fuel injection, where the cylinder heads can be exposed to temperatures exceeding 200 °C during operation. In order to achieve optimal engine temperature during operation, it is necessary to address the impact of individual alloying elements on thermal conductivity of newly developed alloys. The impact of varying Zr addition in the AlSi5Cu2Mg alloy on thermal conductivity in as-cast and heat-treated state is shown in When evaluating the thermal conductivity of experimental alloys in the as-cast state, it was found that the presence of Zr resulted in a 6 to 11% (depending on Zr addition) decrease in thermal conductivity in all variants. From the thermal conductivity point of view, Zr acts as an “impurity”, because the addition of any other element to the alloy has an effect on the decrease in thermal conductivity. Thermal energy in alloys is transferred due to the vibration of atoms and free  electron movement. Excited atoms in one section works to excite and vibrate adjacent atoms. This motion (or kinetic energy) allows heat to move through the alloy. Any alloying element will affect the free electron path and the vibrational state of the surrounding atoms, resulting in a change in thermal conductivity ,33. ThisAfter T7 heat treatment, thermal conductivity of alloy without Zr addition increased by 18%. Depending on the changing Zr addition, thermal conductivity increased by 22 to 33% in Zr-alloyed experimental alloys. However, it should be noted that the addition of Zr in any amount did not have a significant effect on the change in thermal conductivity after heat treatment compared to the alloy without Zr. Thus, it can be stated that the addition of Zr in the AlSi5Cu2Mg alloy did not have a negative effect on the significant reduction in thermal conductivity. This is why it is possible to use such Zr-containing alloy in the development of a new alloy intended for the automotive industry . Thermal conductivity of the experimental alloys in the over-aged T7 state was probably improved due to the formation of fine, uniformly distributed precipitates, which depleted the matrix of solute . SpheroiDepending on Zr addition, the microstructure of experimental alloys in the as-cast state a–e consiAfter T7 heat treatment, dissolution of Cu- and Mg-rich intermetallic phases occurred during solution heat treatment, further creating small, more uniformly dispersed precipitate particles. This was the reason for the significant increase in mechanical properties see a–e. FormZr-rich intermetallic phases were observed in shape of separated thicker needles a or as a15(FeMn)3Si2 with morphology of segmented skeletal structures . The most important findings are:3Zr crystals. The production of aluminum alloys with Zr addition requires accelerated cooling to provide complete entry of Zr into the solid solution, resulting in the formation of a supersaturated solid solution, which subsequently provides an increase in strength properties.From the results obtained during this experiment, it can be concluded that addition of Zr in the AlSi5Cu2Mg alloy showed a deterioration of the mechanical properties. The decrease in mechanical properties was probably related to the insufficient cooling rate during solidification, which led to the formation of primary Al


Lys, is the most common mitochondrial deletion. While it is associated with several diseases like myopathy, dystonia, and hepatocellular carcinoma, it has also been used as an evolutionary marker. However, the mechanism responsible for its fragility is unclear. In the current study, we show that Endonuclease G, a mitochondrial nuclease responsible for nonspecific cleavage of nuclear DNA during apoptosis, can induce breaks at sequences associated with ‘9 bp deletion’ when it is present on a plasmid or in the mitochondrial genome. Through a series of in vitro and intracellular studies, we show that Endonuclease G binds to G-quadruplex structures formed at the hotspot and induces DNA breaks. Therefore, we uncover a new role for Endonuclease G in generating mtDNA deletions, which depends on the formation of G4 DNA within the mitochondrial genome. In summary, we identify a novel property of Endonuclease G, besides its role in apoptosis and the recently described ‘elimination of paternal mitochondria during fertilisation.Having its genome makes the mitochondrion a unique and semiautonomous organelle within cells. Mammalian mitochondrial DNA (mtDNA) is a double-stranded closed circular molecule of about 16 kb coding for 37 genes. Mutations, including deletions in the mitochondrial genome, can culminate in different human diseases. Mapping the deletion junctions suggests that the breakpoints are generally seen at hotspots. ‘9 bp deletion’ (8271–8281), seen in the intergenic region of cytochrome c oxidase II/tRNA The mitochondria are semiautonomous organelles with their genome. The mammalian mitochondrial genome encodes for 13 respiratory chain proteins, 22 tRNAs and 2 rRNAs . Due to The FRA regions and other frequent chromosomal translocations have been observed in late replicating sites in the nuclear DNA, which are prone to replication fork stalling due to possible, stable secondary structure formation or inadvertent action of structure-specific nucleases . MitochoThe presence of alternate DNA structures like G-quadruplex, triplex, and cruciform structures in the mitochondrial genome has been reported along with their association with different mitochondrial diseases . BreakpoLys is one of the most common deletions in mitochondria  9 bp deletion caused by loss of one copy of the 9 bp repeat sequence (CCCCCTCTA) in the intergenic region of cytochrome c oxidase II (COII)/tRNAchondria . It has chondria . The 9 bchondria . The occ4977, and accumulation of it results in different mitochondrial disorders, including cancer   . Recentlan cells . Previou joining . When re joining . A previ joining .Here, we investigate the mechanism of fragility associated with the most common deletion seen in the mitochondrial genome. The putative role of non-B DNA structures and the mechanism of DNA breakage associated with the formation of mtDNA deletions are extensively studied. We report that the region associated with 9 bp deletion can fold into a G-quadruplex DNA structure using various biochemical and ex vivo methods. For the first time, we show that Endonuclease G can specifically bind to the G-quadruplex structure and cleave the mtDNA. Finally, the joining of the broken DNA could lead to the formation of ‘9 bp deletion’ seen in human mitochondria.Previously, different non-B DNA motifs, including G4 DNA motifs have been reported in the mitochondrial genome . It was To investigate whether mitochondrial Region I can indeed form G-quadruplex structures, oligomeric DNA containing the G-rich region referred to as ‘G1’ and complementary C strand (C1) were synthesised . RadiolaDMS protection assay was performed to understand the precise base pairing of guanine nucleotides involved in the G4 plates compared to the ones within connecting loops that facilitate DNA strand folding and, thus, G-quadruplex formation . DMS met+ (+ (with no or little G-quadruplex stabilising properties) or Na+ (with limited stabilising properties) , which could fold into a cruciform DNA, was also present adjacent to the G4 motif. Thus, that was also part of pDI1 . FurtherFormation of G4 DNA on a plasmid or mitochondrial genome could result in single-strandedness at complementary and adjacent regions corresponding to the G4 motif. Sodium bisulphite modification assay was performed on pDI1 to check the single-strandedness in the complementary C strand of Region I. Sodium bisulphite deaminates cytosine resulting in uracil, when present on single-stranded DNA . This chFurther, to investigate the existence of G4 DNA structures in the mitochondrial genome, a sodium bisulphite modification assay was performed after extracting the mitochondrial DNA from human cells  under non-denaturing conditions. Results showed that out of 59 DNA molecules analysed following cloning and sequencing, 52 showed C→T conversions to varying extents . CumulatThe previous studies established that BG4 antibody can bind to G-quadruplexes formed in DNA and RNA . To studTo examine whether BG4 can indeed bind to mitochondrial G-quadruplexes, in vitro binding of purified BG4 to oligomers harbouring the G-quadruplex region was performed . ResultsTo investigate whether BG4 can bind to G-quadruplex DNA present in the mitochondrial genome, the mito IP-qPCR strategy was employed . BrieflyThus, our results suggest that Region I and other G4 motifs in the human mitochondrial genome could fold into G-quadruplex DNA. However, based on bisulphite DNA sequencing from Region I, the structure may not be formed in all DNA molecules, and the frequency may be <25%.To elucidate the mechanism responsible for cleavage at mitochondrial Region I, the involvement of mitochondrial nucleases was considered. To test this, the mitochondrial extract was isolated from the rat tissues (testes and spleen) and used for cleavage assay. The extract was confirmed to be mitochondrial in nature and was tested for cross-contamination by western blotting using the anti-cytochrome C , anti-PCNA (nuclear or cytosolic marker), and anti-Actin (loading control) . PlasmidFor deciphering whether the position of cleavage observed was indeed at Region I, primer extension assay was carried out on pDI1 and its mutant  partially rescued the sensitivity of P1 nuclease .Although Endonuclease G was thought to be localised within the mitochondrial intermembrane space, its proximity to the mitochondrial matrix was supported by multiple experimental evidence suggesting a direct interaction of Endonuclease G with mtDNA . TherefoBased on the present and previous studies , we prop3+N1-7G3+N1-7G3+N1-7G3+). G-quadruplexes with three-plate are considered energetically stable  is the most well-characterized DNA repair pathway in mitochondria, while it is generally believed that nucleotide excision repair is absent . HomologIn a recent study, a replication slippage-mediated mechanism was attributed to a common 4977 deletion where in frequent replication fork stalling at the junction of the common deletion was observed . While tA balance between the formation and resolution of G4 DNA within the mitochondrial genome is essential. The mitochondrial genes are transcribed as a single polycistronic mRNA  and therHence, our study suggests a novel mechanism that explains fragility in mitochondria, dependent on the structure-selective nuclease activity of Endonuclease G on G-quadruplex DNA. The process could be controlled by cellular stress conditions, which may regulate the release of Endonuclease G to the mitochondrial matrix; however, this requires further extensive investigation. Although we have focused on a single example of ‘9 bp deletion’ often seen in mitochondria, it is very much possible that it may be a general mechanism and may help in explaining several other deletions seen in mitochondria-associated human disorders.Chemicals and reagents used in the present study were purchased from Millipore-Sigma (USA), SRL (India), Himedia (India), and Amresco (USA). Restriction enzymes and other DNA-modifying enzymes were obtained from New England Biolabs (USA). Culture media was from Sera Laboratory International Limited (UK), Lonza (UK). Fetal bovine serum and PenStrep were from Gibco BRL (USA). Radioisotope-labelled nucleotides were from BRIT (India). Antibodies were purchased from Abcam (UK), Santa Cruz Biotechnology (USA), BD (USA), Cell Signaling Technology (USA) and Calbiochem (USA). Oligomeric DNA was purchased from Juniper Life Sciences (India) and Medauxin (India).The oligomers used in this study are listed in 2 as described before , HCT116 (human colon cancer), MEF (mouse embryonic fibroblast) and HEK 293T  were purchased from National Centre for Cell Science, Pune, India. Nalm6 and Reh cells were from Dr M. R. Lieber (USA), and Rho(0) cells were from J. Neuzil . The identity of all these cell lines was confirmed by performing STR profiling . All the cell lines were found to be free of mycoplasma contamination. The Mycoplasma test was conducted in the laboratory using the MYCOseq kit  and further validated by DNA labs India, Hyderabad, India. Cells were cultured in RPMI1640 or MEM medium supplemented with 10–15% fetal bovine serum (FBS), 100 μg/ml Penicillin, and 100 μg/ml streptomycin and incubated at 37 °C in a humidified atmosphere containing 5% COd before . Rho(0) d before .Rattus norvegicus) 4–6 weeks old were purchased from the Central animal facility, Indian Institute of Science (IISc), Bangalore, India, and maintained as per the guidelines of the animal ethical committee in accordance with Indian National Law on animal care and use (CAF/Ethics/526/2016).Male Wistar rats (shRNA used in the study (TRCN0000039643) was purchased from the Resource Center at Division of Biological Science, IISc, Bangalore (funded by DBT: BT/PR4982/AGR/36/718/2012) Indian Institute of Science, Bangalore (India).Mitochondrial DNA isolated from Nalm6 cells was amplified using primers specific for Region I (VKK11 and VKK12). The PCR products were purified, blunted and ligated into the EcoRV site of pBlueScript SK(+) to obtain the wild-type plasmid for mitochondrial Region I (pDI1). Mutant plasmids for mitochondrial Region I were generated by PCR-mediated site-directed mutagenesis using pDI1 as a template described before  using thThe endonuclease G coding sequence was amplified using primers VKK21 and VKK22, which were designed with BamHI and NotI restriction sites, respectively, at flanking regions. PCR product was gel purified and cloned into the EcoRV site of pBS SK +cloning vector. For the generation of pRBK2, the coding DNA sequence was digested from the cloning vector using BamHI and NotI enzymes and cloned into the same site (BamHI and NotI) of pET28a+. Mutant plasmid for Endonuclease G was generated by PCR mediated site-directed mutagenesis as described before  using thEscherichia coli was cultured in 500 ml of Luria broth  for 18 h at 37 °C. Isolation of plasmid DNA was performed by the standard alkaline lysis method. It was then purified and precipitated, as described previously , 1% SDS and 0.5 mg/ml Proteinase K to remove denatured proteins. The resulting lysate was deproteinised by phenol-chloroform extraction. The mitochondrial DNA was precipitated with 3 M Sodium acetate (pH 5.2) and chilled with 100% ethanol. The purity of the mitochondrial DNA preparation was evaluated by PCR amplification with nuclear and mitochondrial DNA-specific primers.Mitochondria were isolated from Nalm6 cells as described before . Cells w2, 0.2% Triton X-100, 2 mM of EGTA, 2 mM of EDTA, 1 mM of DTT and 10% glycerol] by mixing (30 min at 4 °C) along with protease inhibitors PMSF (1 mM), aprotinin (1 μg/ml), pepstatin (1 μg/ml) and leupeptin (1 μg/ml). The mitochondrial extract was centrifuged at 12,000 rpm (5 min), and the supernatant  was aliquoted, snap-frozen and stored at −80 °C till use.Mitochondrial protein extracts were prepared following the isolation of mitochondria based on differential centrifugation as described . Testes Mitochondrial protein extracts were also prepared from rat tissues as per the manufacturer’s instructions using a mitochondrial extraction kit . The purity of the mitochondrial extracts prepared was checked by immunoblotting using the nuclear and mitochondrial specific markers.7 HeLa cells were treated with 25 µM Menadione and incubated for 2 hr at 37 °C. The mitochondrial pellet was first sonicated after incubation in hypotonic buffer 10 mM HEPES-KOH pH7.6, 100 mM NaCl, 10 mM Magnesium acetate and 1 mM phenylmethyl sulfonyl fluoride (PMSF) for 15 min. The same buffer containing 500 mM NaCl was added and incubated for another 15 min and then sonicated for 10 min at 37% duty per cycle. The samples were then separated into membrane and soluble fractions by ultracentrifugation at 100,000 g (TLA 110 rotor) for 30 min at 4 °C in OptimaTM TLX table-top ultracentrifuge (Beckman-Coulter)  . The supCoulter) .32P]-ATP in the presence of 1 U of T4 Polynucleotide kinase in a buffer containing 20 mM Tris-acetate [pH 7.9], 10 mM magnesium acetate, 50 mM potassium acetate and 1 mM DTT at 37 °C for 1 h. The radiolabelled oligomers were purified on a Sephadex G25 column and stored at –20 °C until further use (The 5’ end labelling of oligomeric substrates was done using γeLife. Your revised article has been evaluated by Jessica Tyler (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Unleashing a Novel Function of Endonuclease G in Mitochondrial Genome Instability" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:The manuscript makes interesting observations that should advance the field of mitochondrial DNA metabolism and will be off interest for researchers in the field of mitochondrial genome stability and mitochondrial diseases.The revised manuscript is much improved, although there are number of points that should be addressed in a final revision.Essential revisions:1) The manuscript still requires some language editing.2) Page 6, line 33: Overstatement, there are some pause site w/o KCl; change to significantly fewer sites and less pausing.3) Figure 2 D: Primer DI12 is too inefficient to be meaningful. A new primer would be needed to make the point. The question is whether these data are needed in light of the mutant control?4) Page 10, line 24-27 and Figure 6E: Make an additional mutant to test the prediction of the origin of the second remaining band.5) Some points concerning the localisation of the protein in the mitochondria sub-fractions and its proposed recruitment to the matrix are still not convincing (please see specific comments below). There are enough novel data to justify its publication even without including the above-mentioned aspects, or if they are only presented as preliminary data to propose a hypothesis. The discussion should be amended to reflect that.• Line 372: The fact that EndoG is present in mitochondria was already documented by several papers. It is not clear where the novelty is in this Results section. Furthermore, the title mentions TFAM as an inner membrane protein, which is not the case. As mentioned in the text, although TFAM is found associated with the membrane, it is a matrix protein. The low level of co-localisation and the limited resolution of the microscope used make do not allow to conclude much beyond the already known presence of Endo G in mitochondria. More detailed localisation studies would require electron microscopy or super resolution microscopy.• Line 396: Based on the same arguments used above, the results described in the first paragraph of this section do not add much. The resolution of the images does not allow to conclude the presence of Endo G at G-quadruplexes through the co-localisation with the BG4 antibody. However, the PCR experiments provide evidence for that.• The sub-mitochondrial fractionation described does not correspond to the references cited. In both papers cited there is a first step is a hypotonic shock that would yield a pellet of mitoplasts that can then be separated into inner membrane and matrix fractions. Here the fractionation is directly done in high salt and, to my understanding there is no an intermediate step allowing to get rid of the intermembrane fraction or the outer membrane.There is all along an ambiguity regarding the localisation of EndoG. As mentioned by the authors the protein is normally found in the intermembrane space, therefore, the majority of it should be found in the soluble fraction with their fractionation protocol or lost if they use the hypotonic one. However, they find a substantial fraction in the membrane  fraction and this fraction does not decrease upon treatment while the soluble does. The question is then, where does the newly soluble protein come from?eLife. Your revised article has been evaluated by Jessica Tyler (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Unleashing a Novel Function of Endonuclease G in Mitochondrial Genome Instability" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:1) There is still a contradiction in the section describing the localisation of EndoG within the mitochondria. The title of that section (page 11) is "Endonuclease G is expressed in mitochondria and colocalises with inner mitochondria membrane protein TFAM within the cells". However, based on the experiments described in the text that follows and shown in Figure 8, the authors conclude that it colocalises with TFAM which they define as "a transcription factor known to be present in the mitochondrial matrix".2) As described the protocol for mitochondrial fractionation still does not provide a separation of the inner membrane from the outer one, nor does it separate the intermembrane space from the matrix (unless the description omitted a centrifugation step after the hypotonic shock). If this is the case the authors should be more careful in their statements and mention in the text on page 13, as they did in Figure 10, that what they are looking at is the partial release from the membrane to the soluble fractions. Essential revisions:The manuscript makes interesting observations that should advance the field of mitochondrial DNA metabolism and will be off interest for researchers in the field of mitochondrial genome stability and mitochondrial diseases.The revision that is needed to make this manuscript, as submitted, publishable is too extensive. If the authors decide to address the comments by the additional experimentation needed, they are invited to resubmit a revised manuscript as a new submission that I would be happy to handle as BRE and seek to have the same reviewers evaluate the new submission.Alternatively, the authors may want to consider tightening the manuscript and omit the reconstitution studies making sure that their interpretations are supported by the data and consider alternative mechanisms, when necessary. This more focused version would still contain sufficient new insights and allow the authors to further develop the reconstitution experiments with the required exhaustive separate incubation controls for a future publication.We thank the editors and reviewers for their positive comments and suggestions on our manuscript. We have omitted reconstitution experiments as suggested and tightened the manuscript by reorganizing the figures and text to address all the comments, as detailed below.1) Independent of specific criticism towards individual experiments or experimental tools, the manuscript is difficult to read, poorly assembled and would need a major overhaul in the text and figures. The text requires careful editing, there are many disagreements, and many cases where referrals such as 'its' or 'this' are unclear. The figures should be labeled that the experiments can be understood independent of the legend or method description. A few examples are given here.• Figure 1A: The sequences of C1 and G1 are needed here. Giving more is confusing, as they do not relate to the figure. In Figure S1, all oligo sequences should be listed.As suggested by the reviewer, we have removed the oligomer list of mutants from Figure 1  and moved the mutants to Supplement Figure 1 .• Figure S1A: Define -/+ KCl under gel in legend.The suggested label is mentioned below the gel and added also to Figure legend  and .• Figure S1B: Indicate inverted repeat sequences.As suggested by the reviewer, we have now indicated the inverted repeat sequences .• Figure S3C: Where are the gel data? What is shown here? The description in the legend does not allow to understand the figure, what is the difference between the 3 lanes that are labeled as +.We are extremely sorry for the confusion. The gel data and the quantitation are now presented together in the same figure to avoid confusion . The 3 lanes labelled as + are the triplicate samples, which is now explained in the legend .• Figure S3: Unclear what the Westernblots show in D, F, and H.The western blots in D, F, H confirm the identity of purified proteins in the fractions used . Details are added to the legend .• Figure S5F: Define the lanes. What is loaded in lane 4?In old figure S5F , Lane 1 is marker, lanes 2 and 3 are pull down samples loaded after incubation of secondary antibody and anti-endonuclease G, respectively with mitochondrial extract. Lane 4 is mitochondrial extract.• Figure 4D: Label upper input and lower pull-down, also Figure 2G.As suggested, we have labelled the figures .2) A key problem in the manuscript is that the authors mix their interpretation into the description of the results. A few examples are given here, but the authors should check the entire manuscript and correct this issue. The authors should carefully separate description of the results from their interpretation and refrain from overinterpreting their data . There is no need to overstate the result; exceptions or unexpected results must be discussed and acknowledged. For example:• Figure 2F/G: What happened with CR7? Low threshold value but no PCR product? This is not described in the text.When sequence of the region spanning CR7 was analysed for potential G4 motifs, we observed sequence that may support formation of 2 plate G4 structure. This could explain the observed binding of BG4 during semiquantitative PCR. However, the threshold value was based on real time PCR which is highly sensitive when compared to semiquantitative PCR. This could be the reason for absence of a band in the CR7. This is also described in the text now .• Figure 3D: The upper cleavage site stays the same, unlike what is described in the text.The mitochondrial G-quadruplex region I has the ability to form G-quadruplexes in multiple forms . Mutating two of the sequences will still leave with 3-G stretches and 3 GNG sequences which makes the region, a ligand for Endonuclease G binding and cleavage . This is described in the main text .• Figure S3A, B: It is unclear which band is EndoG. Is the Westernblot aligned with material loaded in slots 4-7 of the protein-stained gel? If so, why does the Westernblot intensity not reflect the staining intensity?We have repeated the experiment and provided a new western blot for showing the presence of Endo G in the purified protein. We have marked the band representing Endo G in the figure. For both Endo G and its mutant, fractions 4 and 5, respectively were used for blotting .• Figure 3F: The Mt extract does only show the major EndoG sites but not the minor sites, which add up to a significant proportion of the products.Incubation with purified Endonuclease G resulted in cleavage at multiple positions across the G4 motif. It may be that multiple molecules of purified Endonuclease G may bind to different forms of G4 DNA resulting in cleavage at different positions. In the case of mitochondrial extract, there may be additional proteins, which can bind to G4 DNA, thus preventing cleavage at multiple sites. This has been now added in Page 11, lines 14-20.• Page 10, line 10: The mutant plasmid still has one cleavage site, cleavage is not abrogated as stated in the text.Although, 2 of the G-stretches are mutated, Region I still can fold into a G-quadruplex, thus one of the cleavage sites is not abrogated. We have corrected the statement in the mentioned paragraph .• Page 10, line 22: Figure 3D is extract not purified EndoG as stated in line 22 of page 10.We apologize for the mistake. We have now corrected the statement .• Figure 4E and F: What is the difference between both experiments? E: extract, F: cells? This could be clarified in the figure and text.We apologise for the confusion. Figure 4E  corresponds to the use of mitochondrial extract and 4F  refers use of cells. The same is included in the text as well .• Figure S5G: The description of the P1 results is not precise. There is clear protection of the C strand by EndoG in the upper third of the ladder.As suggested we have edited the text to match with the results in the Figure  .• Figure 7A requires more description and quantitation. Figure 7 B/E: Why is the DAPI signal so variable? Figure 7G: the difference between lanes 1-3 and 4-6 should become apparent from the figure. For Figure 7G and H, only menadione data are shown, there are no IR data as stated in the text.We have now added description and quantitated figure 10A-D. We have also changed the images in figure 10B/E and the DAPI signal is now comparable between the panels. We apologise for the mistake. As mentioned, figure 10G and H is only for Menadione and not for IR.3) The BG4 antibody is problematic. Page 8, line 22: Why is there a BG4 signal in the cytoplasm in rho (0) cells? Is this also seen in rho (+) cells?BG4 binds to G-quadruplexes of nuclear DNA and RNA in cytoplasm . All the BG4 outside nucleus is not only because of G4 DNA in mitochondria, but also due to RNA G4 in the cytoplasm . We have used mitotracker to indicate the mitochondria. Recently, we have also extensively investigated the specificity of BG4 towards G4 DNA . The BG4 signal in the cytoplasm is also seen in rho (+) cells.Also, the authors may want to discuss in more details image modification via Coste's mask. I can clearly see yellow dots in merged images for rho (0) cells, but somehow they are not converting to the white dotes when the Coste's mask is applied.We have now replaced the images to take care of the reviewer’s comments .Figure 2 and 4: There are many BG4 signals? Do the signals all represent G4 tetraplex DNA? How can the authors ascertain the specificity and usefulness of this reagent?The control in Figure 4A/B needs to be defined. What would be expectation from random overlap in this experiment given that both signals are very abundant?Approximately 7,00,000 canonical G4 forming motifs of 25-35 nt are reported in the human nuclear genome . Besides, recently 2 plate G-quadruplexes and involvement of GNG motifs were reported during G-quadruplex structure formation . If one considers all these into account, ~2% of the genome may support the formation of G-quadruplexes in human genome. Thus, the number of BG4 signals can be justified in the nucleus. Within mitochondria 5 canonical G4 motifs  and >200 number of 2 plate G-quadruplexes  have been reported. Considering that several mtDNA are present in one mitochondrion and several mitochondria are present in one cell, impact can be several fold. In addition to that RNA G4 structures have also been reported in cytoplasm .The specificity and usefulness of the reagent has been extensively shown in the previous studies 4) The microscopy approach is less informative and overinterpreted. The level of resolution obtained with the confocal microscope approach used does not allow a conclusion on the localization beyond saying that the protein is present in the mitochondria and is not suitable for distinguishing the different mitochondrial compartments.We agree to the reviewer’s comments that the conclusion on localization based on microscopy approach is not preferred, and thus apart from microscopy we have used the fractionation of mitochondrial compartments to support the data .As for the fractionation procedure used, I am not sure that it allows the separation of the matrix fraction from the intermembrane compartment. Furthermore, the description of the results displayed is not clear and there seem to be some mistakes in the labelling of the figures, making it harder to draw clear conclusions.The fractionation procedure has been used as described in the previous literature . We apologise for the mistakes in labelling and now it has been corrected and presented .5) The mutant substrates are an excellent control for testing the G4 tetraplex hypothesis. What is the experimental evidence that these mutant oligonucleotides do not form tetraplexes?The mutant substrates have been used for the study to understand their mobility along with the wild type oligomers in the gel shift assays . Apart from this, circular dichroism was performed and shown previously for this region and its mutants that they do not fold into G4 DNA . Besides, we have also investigated the same region (with and without mutation) after cloning in a plasmid .6) The attempt to reconstruct the deletion formation in vitro is welcomed. However, these experiments lack a crucial control. In Figure 5B, there is product in the negative control? This raises the concern that bands labeled MMEJ products are PCR artifacts. The authors must conduct a separate incubation control by incubating both oligos separately with extracts, extract the DNA, mix, and then perform PCR detection to ascertain that bands labeled as products are formed during the extract incubation and not PCR artifacts. The recombination literature is replete with this artifact, and it must be eliminated here.The separate incubation controls are also needed for the experiments with mt plasmid DNA and mitochondrial genomics DNA in Figure 5F and Figure S6.In their exploration of the mechanisms leading to deletions, the data presented supports their model invoking MMEJ, at least for the in vitro system as shown by the sequencing of the deletions obtained either with the plasmid or with mitochondrial DNA. When mitochondrial DNA was used, however, it would seem that mutations in that region accumulate but they are not associated with deletions, which are proportionally less abundant than in the plasmid system. This finding merits more discussion.The authors hypothesize that MMEJ repair of the DNA break caused by the endonuclease G produces the 9 bp-deletion. If this is the case, why just one out of four repeats is always deleted? Further, the authors may want to tone down their MMEJ model, since no direct evidence for this mechanism is provided. Other mechanisms, such as nick-repair can potentially lead to the same outcome.As discussed above, based on the suggestion by editors and reviewers we have now removed reconstitution studies from the manuscript. All suggested controls will be performed and a separate manuscript will be submitted at a later timepoint. We sincerely thank the editors for the constructive criticism and for the suggestion.7) Page 13, lines 20/21: Give the exact numbers for 'few' and 'many others'.We have now removed this figure from the manuscript completely and edited the text accordingly.8) The EndoG nuclease experiments and conclusion that EndoG is responsible for the observed cleavages, would be strengthened by purifying a catalytic mutant of EndoG and showing dependance of cleavage on the EndoG active site. This is a standard control for the nuclease field.We thank the editor and reviewer for the suggestion. We have now purified the catalytic mutant of Endo G and shown the cleavage pattern when the mutant protein is used .9) The discussion is not well organized and should include an evaluation what the possible physiological function of the observation of EndoG release upon oxidative stress and deletion formation might be. Are these off-pathway accidents?As suggested, we have now reorganized the discussion. The observation that EndoG is released into matrix upon stress is new in the field. Although our data suggest that it may play a role in deletion formation, further studies are required to demonstrate this .Is there a role of EndoG on G4 RNA tetraplexes? This refers also to the point about the BG4 antibody; does it recognize G4 RNA?Previously, it has been reported that BG4 binds to G4 RNA . However, affinity of Endo G on G4 RNA tetraplexes is yet to be investigated. A detailed study is required before we can comment on this aspect.The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:The manuscript makes interesting observations that should advance the field of mitochondrial DNA metabolism and will be off interest for researchers in the field of mitochondrial genome stability and mitochondrial diseases.The revised manuscript is much improved, although there are number of points that should be addressed in a final revision.We thank the editors for their overall positive comments on our manuscript. Based on their suggestions following changes have been incorporated into the manuscript as detailed below.Essential revisions:1) The manuscript still requires some language editing.We have gone through the manuscript and made additional language corrections as suggested.2) Page 6, line 33: Overstatement, there are some pause site w/o KCl; change to significantly fewer sites and less pausing.As suggested, we have modified the text .3) Figure 2 D: Primer DI12 is too inefficient to be meaningful. A new primer would be needed to make the point. The question is whether these data are needed in light of the mutant control?As rightly pointed out by the reviewer, the efficacy of primer (DI12) binding is inefficient, and this data does not add up significantly to the overall content of Figure 2. Thus, we have removed the panel from figure 2 and reorganised the rest of the figure .4) Page 10, line 24-27 and Figure 6E: Make an additional mutant to test the prediction of the origin of the second remaining band.As suggested by the reviewer, we have now generated a new mutant to test the predicted basis of the second band and presented it as Figure 6-supplement figure 1. Results showed that upon generation of additional mutants, the second band, due to cleavage, disappeared. This further confirms the involvement of G4 DNA during the observed nicking .5) Some points concerning the localisation of the protein in the mitochondria sub-fractions and its proposed recruitment to the matrix are still not convincing (please see specific comments below). There are enough novel data to justify its publication even without including the above-mentioned aspects, or if they are only presented as preliminary data to propose a hypothesis. The discussion should be amended to reflect that.We agree with the reviewer, and as suggested, the immunolocalisation data showing the recruitment of Endonuclease G to the matrix is now removed from Figure 10. However, we have maintained sub-fractionation data as a preliminary observation .• Line 372: The fact that EndoG is present in mitochondria was already documented by several papers. It is not clear where the novelty is in this Results section. Furthermore, the title mentions TFAM as an inner membrane protein, which is not the case. As mentioned in the text, although TFAM is found associated with the membrane, it is a matrix protein. The low level of co-localisation and the limited resolution of the microscope used make do not allow to conclude much beyond the already known presence of Endo G in mitochondria. More detailed localisation studies would require electron microscopy or super resolution microscopy.We agree with the reviewer’s comments that the conclusion on sub-localisation based on the standard microscopy technique may not be sufficient to make the point. Thus, we have removed the immunofluorescence figures related to the localisation of EndoG to different mitochondrial compartments.• Line 396: Based on the same arguments used above, the results described in the first paragraph of this section do not add much. The resolution of the images does not allow to conclude the presence of Endo G at G-quadruplexes through the co-localisation with the BG4 antibody. However, the PCR experiments provide evidence for that.We thank the reviewer for their comment. We agree that the results described do not add much and the presence of BG4 cannot be concluded based on only colocalization with BG4. However, as stated, since we have other experiments (PCR-based ChIP) to evaluate the same question, we have maintained the data in the manuscript .• The sub-mitochondrial fractionation described does not correspond to the references cited. In both papers cited there is a first step is a hypotonic shock that would yield a pellet of mitoplasts that can then be separated into inner membrane and matrix fractions. Here the fractionation is directly done in high salt and, to my understanding there is no an intermediate step allowing to get rid of the intermembrane fraction or the outer membrane.We apologise for not providing a detailed protocol. As mentioned in Sinha et al., we first performed the hypotonic shock to yield mitoplasts and separated them into inner membrane and matrix fractions . Accordingly, we have rewritten the section by providing sufficient detail to avoid ambiguity .There is all along an ambiguity regarding the localisation of EndoG. As mentioned by the authors the protein is normally found in the intermembrane space, therefore, the majority of it should be found in the soluble fraction with their fractionation protocol or lost if they use the hypotonic one. However, they find a substantial fraction in the membrane  fraction and this fraction does not decrease upon treatment while the soluble does. The question is then, where does the newly soluble protein come from?We agree with the reviewer that there has been ambiguity in the sublocalization of EndoG. While there are few papers suggesting the localization to inner membrane :1147-55,Ishihara and Shimamoto (2006) JBC; 281, (10): 6726 –6733) others suggest its presence in the inner membrane space :394-9, Davies et al., 2003 Nucleic acids Res. 31(4): 1364–1373). With the protocol that we have used (the hypotonic method), if it was present only in the inner membrane space, Endo G would have been completely lost. However, further investigation is required to comment on this aspect.We apologise for the ambiguity of quantification. We have now re-quantified the western blots and presented it as Figure 10B.The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:1) There is still a contradiction in the section describing the localisation of EndoG within the mitochondria. The title of that section (page 11) is "Endonuclease G is expressed in mitochondria and colocalises with inner mitochondria membrane protein TFAM within the cells". However, based on the experiments described in the text that follows and shown in Figure 8, the authors conclude that it colocalises with TFAM which they define as "a transcription factor known to be present in the mitochondrial matrix".We thank the reviewer for pointing out this and apologize for the error. We have now corrected the title section on page 12, lines 1, 2.2) As described the protocol for mitochondrial fractionation still does not provide a separation of the inner membrane from the outer one, nor does it separate the intermembrane space from the matrix (unless the description omitted a centrifugation step after the hypotonic shock). If this is the case the authors should be more careful in their statements and mention in the text on page 13, as they did in Figure 10, that what they are looking at is the partial release from the membrane to the soluble fractions.Thank you for the suggestion. We have now edited the text to avoid the discrepancy .