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. 2012 Mar 16;287(12):9613-22.
doi: 10.1074/jbc.M111.331728. Epub 2012 Feb 2.

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  1 , Youngjin Cho, In-Young Yang, Jun-ichi Akagi, Eiji Ohashi, Satoshi Tateishi, Niels de Wind, Fumio Hanaoka, Haruo Ohmori, Masaaki Moriya
Affiliations

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 et al. J Biol Chem. .

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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Figures

FIGURE 1.
FIGURE 1.
Preparation of BPDE-dG-bearing plasmid and oligonucleotide probes used for analysis. A, vector-employed, BPDE-dG insertion site (open circle) is located between BsaI and BsmBI. Py, mouse polyoma virus; ori, replication origin; amp, ampicillin resistance gene; blaS, blasticidin S resistance gene. B, preparation of gapped plasmid, B with gray shading represents BPDE-dG. Note three mismatches at 5′-BCC/5′-UCU. A, C, G, and T probes determine a TLS event targeting BPDE-dG, whereas L and R probes determine the presence of the inserted modified oligonucleotide.
FIGURE 2.
FIGURE 2.
Relative efficiency and targeted coding specificity of translesion DNA synthesis across BPDE-dG (A and C) and H-ϵdC (B). A and B, TKO, Polk−/− Polh−/− Poli−/− triple-gene knockout MEF. k/i, Polk−/− Poli−/− double-gene knockout MEF; h/i, Polh−/− Poli−/− double-gene knockout MEF. A value for wild-type MEF is set to 100%. Coding specificity is color-coded. Red, BPDE-dG (G*) or H-ϵdC (C*) → T; blue, G* or C* → A; purple, G* or C* → C; green, G* or C* → G; black, others. Detailed numbers, %, and statistics are reported in supplemental Tables S1 and S2.
FIGURE 3.
FIGURE 3.
REV7-mediated interaction between REV1 and REV3 in yeast. A, principle of the assay. GAL4 BD-fused bait protein (REV3) and nontagged REV7 are expressed from pBridge. The expression of REV7 from the promoter, PMet25, is repressed in the presence of methionine in medium, and hence the gene (REV7) cloned in the cloning site II is not expressed. It becomes active in the absence of methionine and expresses the gene. The GAL4 AD-fused prey protein (REV1) is expressed constitutively from pGADT7. When three gene products form a heterotrimer (panel a), a marker gene is expressed. The absence of a mediator protein (REV7) (panel b) or overexpression of a mediator protein (panel c) causes a failure of the formation of a heterotrimer, and a marker gene is not expressed. B, formation of heterotrimer as judged by the growth of a yeast host on selective media. The numbers correspond to those in Table 2, showing the combination of genes tested. Panels I, II, and III are two-nutrient (Leu and Trp), four-nutrient (Leu, Trp, Ade, and His), and five-nutrient (Leu, Trp, Ade, His, and Met) dropout media, respectively.
FIGURE 4.
FIGURE 4.
REV7-mediated interaction between REV1 and REV3 in mammalian cells. The following genes were expressed in the monkey kidney cell line, COS7. Square, GAL4 BD-REV3(1776–2003) fusion, VP16-AD-REV1(1141–1251) fusion, and full-length REV7; circle, BD-REV3(1776–2003) and AD-REV1(1141–1251); triangle, BD-REV3(1776–2003), AD and REV7. Culture media were collected from day 1 through day 6 following transfection and assayed for BD-REV3(1776–2003):AD-REV1(1141–1251) interaction by a Great EscAPe SEAP chemiluminescence detection kit (Clontech). Similar results were obtained in several experiments conducted under different conditions. Refer to the legend to Fig. 3 for the principle of assay and “Experimental Procedures” for details.
FIGURE 5.
FIGURE 5.
Model for TLS across BPDE-dG and recruitment of pol ζ to a replication-stalled site. Following the insertion of a nucleotide (N) opposite BPDE-dG (red triangle), extension from this newly created terminus requires pol ζ when N is incorrect A, T, or G. On the other hand, when N is correct, C (the extension) does not necessarily depend on pol ζ. Pol κ might conduct this extension. Pol ζ is recruited by REV1 through the interaction between REV1 and REV7, a subunit of pol ζ (thick red arrow). Then REV1 may interact with monoubiquitinated PCNA at its ubiquitin-binding motifs to bring pol ζ to a stalled site. This is the major pol ζ recruitment pathway. A REV1-independent minor pathway (thin red arrow) also exists, but its mechanism is unclear. U, 1, 3, and 7 represent monoubiquitin, REV1, REV3 and REV7, respectively.
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