Quantification of pyrophosphate as a universal approach to determine polymerase activity and assay polymerase inhibitors.
The importance of DNA polymerases in biology and biotechnology, and their recognition as potential therapeutic targets, drives development of methods for deriving kinetic characteristics of polymerases and their propensity to perform polynucleotide synthesis over modified DNA templates. Among various polymerases, translesion synthesis (TLS) polymerases enable cells to avoid the cytotoxic stalling of replicative DNA polymerases at chemotherapy-induced DNA lesions, thereby leading to drug resistance. Identification of TLS inhibitors to overcome drug-resistance necessitates the development of appropriate high-throughput assays. Since polymerase-mediated DNA synthesis involves the release of inorganic pyrophosphate (PPi), we established a universal and fast method for monitoring the progress of DNA polymerases based on the quantification of PPi with a fluorescence-based assay that we coupled to in vitro primer extension reactions. The established assay has a nanomolar detection limit in PPi and enables the evaluation of single nucleotide incorporation and DNA synthesis progression kinetics. The results demonstrated that the developed assay is a reliable method for monitoring TLS and identifying nucleoside and nucleotide-based TLS inhibitors.
['Malvezzi|S|S|', 'Sturla|S J|SJ|', 'Tanasova|M|M|']
D004247:DNA D004259:DNA-Directed DNA Polymerase D011756:Diphosphates D057075:Enzyme Assays D005453:Fluorescence D009711:Nucleotides D048229:Sulfolobus solfataricus
TLS inhibitors DNA polymerase activity Nucleotide analogs Pyrophosphate detection Translesion DNA synthesis