Functions of base selection step in human DNA polymerase α

Recent studies have revealed that the base selection step of DNA polymerases (pol) plays a role in prevention of DNA replication errors. We investigated whether base selection is required for the DNA replication fidelity of pol α and genomic stability in human cells. We introduced an Leu864 to Phe substitution (L864F) into human pol α and performed an in vitro LacZα forward mutation assay. Our results showed that the overall mutation rate was increased by 180-fold as compared to that of the wild-type. Furthermore, steady state kinetics analyses consistently showed that L864F pol α had a decreased discrimination ability between correct and incorrect nucleotide incorporation, as well as between matched and mismatched primer termini. L864F pol α also exhibited increased translesion activity over the abasic, etheno-A, O⁴-methyl-T, and O⁶-methyl-G sites. In addition, our steady state kinetics analyses supported the finding of increased translesion activity of L864F pol α over O⁶-methyl-G. We also established stable clones transfected with pola1L864F utilizing the human cancer cell line HCT116. Using the HPRT gene as a reporter, the spontaneous mutation rate of pola1L864F cells was determined to be 2.4-fold greater than that of wild-type cells. Mutation assays were also carried out using cells transiently transfected with the wild-type or pola1L864F, and increased mutant frequencies were observed in pola1L864F cells under both spontaneous and methyl methanesulfonate-induced conditions. Together, our results indicate that the base selection step in human pol α functions to prevent DNA replication errors and maintain genomic integrity in HCT116 cells.