DNA polymerases are specialized for replication, repair and to tolerate DNA damage. Anomalies in proofreading domains of pol and pol are associated with cancer but the roles of mutations in Y-family DNA polymerases such as pol are less well understood. Y-family DNA polymerases are specialized in translesion synthesis, which allows bypassing DNA damage sites that could block the normal progression of the replication forks. Previous studies have shown the ability of pol to bypass minor groove DNA adducts but inhibition by major groove adducts. Hundreds of single nucleotide polymorphisms (SNPs) in pol associated with different types of cancer were found by database searches. The m₄value, a metric of the degree of coupling between one ionizable amino acid and its neighbors obtained from POOL and THEMATICS, was used to identify which protein SNPs are likely to impact the polymerase activity when compared with wild type (WT) pol. With the use of bioinformatics tools SIFT, PolyPhen, and FATHMM, we found that most of these variants are predicted to be deleterious and could affect the function of the protein. With this information, we have identified 18 pol variants that are most likely to affect the polymerase function. We assessed the overall stability of WT pol and pol variants using thermal shift assays. We found that many of the variants representing these cancer-associated SNPs showed similar melting temperatures as WT pol (42.5 °C ± 0.3 °C) while two of 18 selected, R298C (33.9 °C ± 0.6 °C) and L383F (33.8 °C ± 0.5 °C) showed lower thermal stability. All 18 variants showed a similar trend with WT pol, which is a slight increase in the melting temperature in the presence of primer: template DNA. With the addition of the correct nucleotide, melting temperatures were increased by 10-15 °C, whereas with incorrect nucleotide, the temperatures were increased by only 4-7 °C for all proteins. Protein variants in the presence of DNA templates with a minor groove N²ffdG adduct showed increased thermal stability compared to WT pol, supporting the argument that these pol variants can bypass DNA damage lesions. Furthermore, primer extension assays conducted with these cancer-associated pol variants showed that variants R48I, T102A, H142Y, V177L, and N338S can bypass N²ffdG DNA adducts. Their extension ability in the presence of correct and incorrect nucleotides is yet to be determined.

Lakindu Pathira Kankanamge

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