title: Functions of base selection step in human DNA polymerase alpha.
abstract: 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 alpha and genomic stability in human cells. We introduced an Leu864 to Phe substitution (L864F) into human pol alpha and performed an in vitro LacZ alpha 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 alpha had a decreased discrimination ability between correct and incorrect nucleotide incorporation, as well as between matched and mismatched primer termini. L864F pol alpha also exhibited increased translesion activity over the abasic, etheno-A, O(4)-methyl-T, and O(6)-methyl-G sites. In addition, our steady state kinetics analyses supported the finding of increased translesion activity of L864F pol alpha over O(6)-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 alpha functions to prevent DNA replication errors and maintain genomic integrity in HCT116 cells.
authors.affiliation: Division of Molecular Carcinogenesis, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Tanaka
authors.forename: Ke
authors.initials: K
authors.lastname: Cao
authors.forename: Atsuko
authors.initials: A
authors.lastname: Niimi
authors.forename: Siripan
authors.initials: S
authors.lastname: Limsirichaikul
authors.forename: Huang Qin
authors.initials: HQ
authors.lastname: Miao
authors.forename: Noriko
authors.initials: N
authors.lastname: Nakamura
authors.forename: Takashi
authors.initials: T
authors.lastname: Murate
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Hasegawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Takahashi
authors.forename: Motoshi
authors.initials: M
authors.lastname: Suzuki
mesh_terms: D019943:Amino Acid Substitution; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D045325:HCT116 Cells; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D013379:Substrate Specificity
chemical_list: D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D004256:DNA Polymerase I