Functions of base selection step in human DNA polymerase α

Shigeru Tanaka, Ke Cao, Atsuko Niimi, Siripan Limsirichaikul, Huang Qin Miao, Noriko Nakamura, Takashi Murate, Yoshinori Hasegawa, Takashi Takahashi, Motoshi Suzuki

研究成果: Article査読

8 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)534-541
ページ数8
ジャーナルDNA Repair
9
5
DOI
出版ステータスPublished - 04-05-2010
外部発表はい

All Science Journal Classification (ASJC) codes

  • 生化学
  • 分子生物学
  • 細胞生物学

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