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Two-polymerase mechanisms dictate error-free and error-prone translesion DNA synthesis in mammals.

ABSTRACT: DNA replication across blocking lesions occurs by translesion DNA synthesis (TLS), involving a multitude of mutagenic DNA polymerases that operate to protect the mammalian genome. Using a quantitative TLS assay, we identified three main classes of TLS in human cells: two rapid and error-free, and the third slow and error-prone. A single gene, REV3L, encoding the catalytic subunit of DNA polymerase zeta (pol zeta), was found to have a pivotal role in TLS, being involved in TLS across all lesions examined, except for a TT cyclobutane dimer. Genetic epistasis siRNA analysis indicated that discrete two-polymerase combinations with pol zeta dictate error-prone or error-free TLS across the same lesion. These results highlight the central role of pol zeta in both error-prone and error-free TLS in mammalian cells, and show that bypass of a single lesion may involve at least three different DNA polymerases, operating in different two-polymerase combinations.

SUBMITTER: Shachar S

PROVIDER: S-EPMC2646147 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Two-polymerase mechanisms dictate error-free and error-prone translesion DNA synthesis in mammals.

Shachar Sigal S Ziv Omer O Avkin Sharon S Adar Sheera S Wittschieben John J Reissner Thomas T Chaney Stephen S Friedberg Errol C EC Wang Zhigang Z Carell Thomas T Geacintov Nicholas N Livneh Zvi Z

The EMBO journal 20090115 4

DNA replication across blocking lesions occurs by translesion DNA synthesis (TLS), involving a multitude of mutagenic DNA polymerases that operate to protect the mammalian genome. Using a quantitative TLS assay, we identified three main classes of TLS in human cells: two rapid and error-free, and the third slow and error-prone. A single gene, REV3L, encoding the catalytic subunit of DNA polymerase zeta (pol zeta), was found to have a pivotal role in TLS, being involved in TLS across all lesions ...[more]

PMID: 19153606

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Project description:DNA polymerase (pol) ? is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol ? can bypass various DNA lesions, e.g., N(2)-ethyl(Et)G, O(6)-methyl(Me)G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol ? on its TLS properties, using the recombinant pol ? (residues 1-445) proteins and DNA templates containing a G, N(2)-EtG, O(6)-MeG, 8-oxoG, or abasic site. The ?1-25 variant, which is the N-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg(2+) (but not with Mn(2+)), coinciding with its steady-state kinetic data showing a ?10-fold increase in kcat/Km for nucleotide incorporation opposite templates (only with Mg(2+)). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in kcat/Km for nucleotide incorporation opposite templates either with Mg(2+) or Mn(2+), except for that opposite N(2)-EtG with Mn(2+) (showing a 9-fold increase for dCTP incorporation). The ?1-25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg(2+)), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the ?1-25 variant, was ?7-fold stronger with 0.15 mM Mn(2+) than with Mg(2+). The results indicate that the R96G variation severely impairs most of the Mg(2+)- and Mn(2+)-dependent TLS abilities of pol ?, whereas the ?1-25 variation selectively and substantially enhances the Mg(2+)-dependent TLS capability of pol ?, emphasizing the potential translational importance of these pol ? genetic variations, e.g., individual differences in TLS, mutation, and cancer susceptibility to genotoxic carcinogens.

Dataset Information

Two-polymerase mechanisms dictate error-free and error-prone translesion DNA synthesis in mammals.

Publications

Two-polymerase mechanisms dictate error-free and error-prone translesion DNA synthesis in mammals.

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