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Regulation of error-prone translesion synthesis by Spartan/C1orf124.

ABSTRACT: Translesion synthesis (TLS) employs low fidelity polymerases to replicate past damaged DNA in a potentially error-prone process. Regulatory mechanisms that prevent TLS-associated mutagenesis are unknown; however, our recent studies suggest that the PCNA-binding protein Spartan plays a role in suppression of damage-induced mutagenesis. Here, we show that Spartan negatively regulates error-prone TLS that is dependent on POLD3, the accessory subunit of the replicative DNA polymerase Pol ?. We demonstrate that the putative zinc metalloprotease domain SprT in Spartan directly interacts with POLD3 and contributes to suppression of damage-induced mutagenesis. Depletion of Spartan induces complex formation of POLD3 with Rev1 and the error-prone TLS polymerase Pol ?, and elevates mutagenesis that relies on POLD3, Rev1 and Pol ?. These results suggest that Spartan negatively regulates POLD3 function in Rev1/Pol ?-dependent TLS, revealing a previously unrecognized regulatory step in error-prone TLS.

SUBMITTER: Kim MS

PROVIDER: S-EPMC3561950 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Regulation of error-prone translesion synthesis by Spartan/C1orf124.

Kim Myoung Shin MS Machida Yuka Y Vashisht Ajay A AA Wohlschlegel James A JA Pang Yuan-Ping YP Machida Yuichi J YJ

Nucleic acids research 20121218 3

Translesion synthesis (TLS) employs low fidelity polymerases to replicate past damaged DNA in a potentially error-prone process. Regulatory mechanisms that prevent TLS-associated mutagenesis are unknown; however, our recent studies suggest that the PCNA-binding protein Spartan plays a role in suppression of damage-induced mutagenesis. Here, we show that Spartan negatively regulates error-prone TLS that is dependent on POLD3, the accessory subunit of the replicative DNA polymerase Pol δ. We demon ...[more]

PMID: 23254330

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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

Regulation of error-prone translesion synthesis by Spartan/C1orf124.

Publications

Regulation of error-prone translesion synthesis by Spartan/C1orf124.

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