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

Research output: Contribution to journalArticle

8 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)534-541
Number of pages8
JournalDNA Repair
Volume9
Issue number5
DOIs
Publication statusPublished - 04-05-2010

Fingerprint

DNA-Directed DNA Polymerase
DNA Replication
Assays
DNA
Cells
Methyl Methanesulfonate
Hypoxanthine Phosphoribosyltransferase
Mutation Rate
Kinetics
HCT116 Cells
Substitution reactions
Nucleotides
Genes
Mutation
Genomic Instability
Clone Cells
Cell Line
Neoplasms

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Tanaka, S., Cao, K., Niimi, A., Limsirichaikul, S., Miao, H. Q., Nakamura, N., ... Suzuki, M. (2010). Functions of base selection step in human DNA polymerase α. DNA Repair, 9(5), 534-541. https://doi.org/10.1016/j.dnarep.2010.02.002
Tanaka, Shigeru ; Cao, Ke ; Niimi, Atsuko ; Limsirichaikul, Siripan ; Miao, Huang Qin ; Nakamura, Noriko ; Murate, Takashi ; Hasegawa, Yoshinori ; Takahashi, Takashi ; Suzuki, Motoshi. / Functions of base selection step in human DNA polymerase α. In: DNA Repair. 2010 ; Vol. 9, No. 5. pp. 534-541.
@article{6dd44930abac4a57bda3d88839bc26b9,
title = "Functions of base selection step in human DNA polymerase α",
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 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.",
author = "Shigeru Tanaka and Ke Cao and Atsuko Niimi and Siripan Limsirichaikul and Miao, {Huang Qin} and Noriko Nakamura and Takashi Murate and Yoshinori Hasegawa and Takashi Takahashi and Motoshi Suzuki",
year = "2010",
month = "5",
day = "4",
doi = "10.1016/j.dnarep.2010.02.002",
language = "English",
volume = "9",
pages = "534--541",
journal = "DNA Repair",
issn = "1568-7864",
publisher = "Elsevier",
number = "5",

}

Tanaka, S, Cao, K, Niimi, A, Limsirichaikul, S, Miao, HQ, Nakamura, N, Murate, T, Hasegawa, Y, Takahashi, T & Suzuki, M 2010, 'Functions of base selection step in human DNA polymerase α', DNA Repair, vol. 9, no. 5, pp. 534-541. https://doi.org/10.1016/j.dnarep.2010.02.002

Functions of base selection step in human DNA polymerase α. / Tanaka, Shigeru; Cao, Ke; Niimi, Atsuko; Limsirichaikul, Siripan; Miao, Huang Qin; Nakamura, Noriko; Murate, Takashi; Hasegawa, Yoshinori; Takahashi, Takashi; Suzuki, Motoshi.

In: DNA Repair, Vol. 9, No. 5, 04.05.2010, p. 534-541.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Functions of base selection step in human DNA polymerase α

AU - Tanaka, Shigeru

AU - Cao, Ke

AU - Niimi, Atsuko

AU - Limsirichaikul, Siripan

AU - Miao, Huang Qin

AU - Nakamura, Noriko

AU - Murate, Takashi

AU - Hasegawa, Yoshinori

AU - Takahashi, Takashi

AU - Suzuki, Motoshi

PY - 2010/5/4

Y1 - 2010/5/4

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=77952101773&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77952101773&partnerID=8YFLogxK

U2 - 10.1016/j.dnarep.2010.02.002

DO - 10.1016/j.dnarep.2010.02.002

M3 - Article

C2 - 20202915

AN - SCOPUS:77952101773

VL - 9

SP - 534

EP - 541

JO - DNA Repair

JF - DNA Repair

SN - 1568-7864

IS - 5

ER -

Tanaka S, Cao K, Niimi A, Limsirichaikul S, Miao HQ, Nakamura N et al. Functions of base selection step in human DNA polymerase α. DNA Repair. 2010 May 4;9(5):534-541. https://doi.org/10.1016/j.dnarep.2010.02.002