The vital role of polymerase ζ and REV1 in mutagenic, but not correct, DNA synthesis across Benzo[a]pyrene-dG and recruitment of polymerase ζ by REV1 to replication-stalled site

Keiji Hashimoto, Youngjin Cho, In Young Yang, Jun Ichi Akagi, Eiji Ohashi, Satoshi Tateishi, Niels De Wind, Fumio Hanaoka, Haruo Ohmori, Masaaki Moriya

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

The DNA synthesis across DNA lesions, termed translesion synthesis (TLS), is a complex process influenced by various factors. To investigate this process in mammalian cells, we examined TLS across a benzo[a]pyrene dihydrodiol epoxide-derived dG adduct (BPDE-dG) using a plasmid bearing a single BPDE-dG and genetically engineered mouse embryonic fibroblasts (MEFs). In wild-type MEFs, TLS was extremely miscoding (>90%) with G → T transversions being predominant. Knockout of the Rev1 gene decreased both the TLS efficiency and the miscoding frequency. Knockout of the Rev3L gene, coding for the catalytic subunit of pol ζ, caused even greater decreases in these two TLS parameters; almost all residual TLS were error-free. Thus, REV1 and pol ζ are critical to mutagenic, but not accurate, TLS across BPDE-dG. The introduction of human REV1 cDNA into Rev1 -/- MEFs restored the mutagenic TLS, but a REV1 mutant lacking the C terminus did not. Yeast and mammalian three-hybrid assays revealed that the REV7 subunit of pol ζ mediated the interaction between REV3 and the REV1 C terminus. These results support the hypothesis that REV1 recruits pol ζ through the interaction with REV7. Our results also predict the existence of a minor REV1-independent pol ζ recruitment pathway. Finally, although mutagenic TLS across BPDE-dG largely depends on RAD18, experiments using Polk -/- Polh -/- Poli -/- triple-gene knockout MEFs unexpectedly revealed that another polymerase(s) could insert a nucleotide opposite BPDE-dG. This indicates that a non-Y family polymerase(s) can insert a nucleotide opposite BPDE-dG, but the subsequent extension from miscoding termini depends on REV1-polζ in a RAD18-dependent manner.

Original languageEnglish
Pages (from-to)9613-9622
Number of pages10
JournalJournal of Biological Chemistry
Volume287
Issue number12
DOIs
Publication statusPublished - Mar 16 2012

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Benzo(a)pyrene
Gene Knockout Techniques
DNA
Fibroblasts
Nucleotides
Genes
Two-Hybrid System Techniques
Epoxy Compounds
Knockout Mice
Bearings (structural)
7,8-dihydroxy-9,10-epoxide-7,8,9,10-tetrahydrobenzo(a)pyrene-10-deoxyguanosine
Catalytic Domain
Plasmids
Complementary DNA
Yeast
Assays
Cells

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

The vital role of polymerase ζ and REV1 in mutagenic, but not correct, DNA synthesis across Benzo[a]pyrene-dG and recruitment of polymerase ζ by REV1 to replication-stalled site. / Hashimoto, Keiji; Cho, Youngjin; Yang, In Young; Akagi, Jun Ichi; Ohashi, Eiji; Tateishi, Satoshi; De Wind, Niels; Hanaoka, Fumio; Ohmori, Haruo; Moriya, Masaaki.

In: Journal of Biological Chemistry, Vol. 287, No. 12, 16.03.2012, p. 9613-9622.

Research output: Contribution to journalArticle

Hashimoto, Keiji ; Cho, Youngjin ; Yang, In Young ; Akagi, Jun Ichi ; Ohashi, Eiji ; Tateishi, Satoshi ; De Wind, Niels ; Hanaoka, Fumio ; Ohmori, Haruo ; Moriya, Masaaki. / The vital role of polymerase ζ and REV1 in mutagenic, but not correct, DNA synthesis across Benzo[a]pyrene-dG and recruitment of polymerase ζ by REV1 to replication-stalled site. In: Journal of Biological Chemistry. 2012 ; Vol. 287, No. 12. pp. 9613-9622.
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abstract = "The DNA synthesis across DNA lesions, termed translesion synthesis (TLS), is a complex process influenced by various factors. To investigate this process in mammalian cells, we examined TLS across a benzo[a]pyrene dihydrodiol epoxide-derived dG adduct (BPDE-dG) using a plasmid bearing a single BPDE-dG and genetically engineered mouse embryonic fibroblasts (MEFs). In wild-type MEFs, TLS was extremely miscoding (>90{\%}) with G → T transversions being predominant. Knockout of the Rev1 gene decreased both the TLS efficiency and the miscoding frequency. Knockout of the Rev3L gene, coding for the catalytic subunit of pol ζ, caused even greater decreases in these two TLS parameters; almost all residual TLS were error-free. Thus, REV1 and pol ζ are critical to mutagenic, but not accurate, TLS across BPDE-dG. The introduction of human REV1 cDNA into Rev1 -/- MEFs restored the mutagenic TLS, but a REV1 mutant lacking the C terminus did not. Yeast and mammalian three-hybrid assays revealed that the REV7 subunit of pol ζ mediated the interaction between REV3 and the REV1 C terminus. These results support the hypothesis that REV1 recruits pol ζ through the interaction with REV7. Our results also predict the existence of a minor REV1-independent pol ζ recruitment pathway. Finally, although mutagenic TLS across BPDE-dG largely depends on RAD18, experiments using Polk -/- Polh -/- Poli -/- triple-gene knockout MEFs unexpectedly revealed that another polymerase(s) could insert a nucleotide opposite BPDE-dG. This indicates that a non-Y family polymerase(s) can insert a nucleotide opposite BPDE-dG, but the subsequent extension from miscoding termini depends on REV1-polζ in a RAD18-dependent manner.",
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T1 - The vital role of polymerase ζ and REV1 in mutagenic, but not correct, DNA synthesis across Benzo[a]pyrene-dG and recruitment of polymerase ζ by REV1 to replication-stalled site

AU - Hashimoto, Keiji

AU - Cho, Youngjin

AU - Yang, In Young

AU - Akagi, Jun Ichi

AU - Ohashi, Eiji

AU - Tateishi, Satoshi

AU - De Wind, Niels

AU - Hanaoka, Fumio

AU - Ohmori, Haruo

AU - Moriya, Masaaki

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AB - The DNA synthesis across DNA lesions, termed translesion synthesis (TLS), is a complex process influenced by various factors. To investigate this process in mammalian cells, we examined TLS across a benzo[a]pyrene dihydrodiol epoxide-derived dG adduct (BPDE-dG) using a plasmid bearing a single BPDE-dG and genetically engineered mouse embryonic fibroblasts (MEFs). In wild-type MEFs, TLS was extremely miscoding (>90%) with G → T transversions being predominant. Knockout of the Rev1 gene decreased both the TLS efficiency and the miscoding frequency. Knockout of the Rev3L gene, coding for the catalytic subunit of pol ζ, caused even greater decreases in these two TLS parameters; almost all residual TLS were error-free. Thus, REV1 and pol ζ are critical to mutagenic, but not accurate, TLS across BPDE-dG. The introduction of human REV1 cDNA into Rev1 -/- MEFs restored the mutagenic TLS, but a REV1 mutant lacking the C terminus did not. Yeast and mammalian three-hybrid assays revealed that the REV7 subunit of pol ζ mediated the interaction between REV3 and the REV1 C terminus. These results support the hypothesis that REV1 recruits pol ζ through the interaction with REV7. Our results also predict the existence of a minor REV1-independent pol ζ recruitment pathway. Finally, although mutagenic TLS across BPDE-dG largely depends on RAD18, experiments using Polk -/- Polh -/- Poli -/- triple-gene knockout MEFs unexpectedly revealed that another polymerase(s) could insert a nucleotide opposite BPDE-dG. This indicates that a non-Y family polymerase(s) can insert a nucleotide opposite BPDE-dG, but the subsequent extension from miscoding termini depends on REV1-polζ in a RAD18-dependent manner.

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