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.
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.
Department of Pharmacological Sciences, State University of New York, Stony Brook, New York 11794-8651, USA.
Keiji
K
Hashimoto
Youngjin
Y
Cho
In-Young
IY
Yang
Jun-ichi
J
Akagi
Eiji
E
Ohashi
Satoshi
S
Tateishi
Niels
N
de Wind
Fumio
F
Hanaoka
Haruo
H
Ohmori
Masaaki
M
Moriya
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000031:analogs & derivatives; D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D018736:DNA Adducts / Q000235:genetics; D004249:DNA Damage; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000235:genetics; D006801:Humans; D064246:Mad2 Proteins; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000187:drug effects; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D000099270:Y-Family DNA Polymerases
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D018797:Cell Cycle Proteins; D018736:DNA Adducts; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D009713:Nucleotidyltransferases; D011506:Proteins; C120552:MAD2L2 protein, human; C577699:Mad2l2 protein, mouse; C040060:7,8-dihydroxy-9,10-epoxide-7,8,9,10-tetrahydrobenzo(a)pyrene-10-deoxyguanosine; C425929:DNA polymerase zeta; C429004:REV1 protein, human; D000099270:Y-Family DNA Polymerases; C486862:REV3L protein, mouse; C118285:REV3L protein, human; C496086:Rev1 protein, mouse