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Bypass of a psoralen DNA interstrand cross-link by DNA polymerases ?, ?, and ? in vitro.

ABSTRACT: Repair of DNA interstrand cross-links in mammalian cells involves several biochemically distinctive processes, including the release of one of the cross-linked strands and translesion DNA synthesis (TLS). In this report, we investigated the in vitro TLS activity of a psoralen DNA interstrand cross-link by three DNA repair polymerases, DNA polymerases ?, ?, and ?. DNA polymerase ? is capable of bypassing a psoralen cross-link with a low efficiency. Cell extracts prepared from DNA polymerase ? knockout mouse embryonic fibroblasts showed a reduced bypass activity of the psoralen cross-link, and purified DNA polymerase ? restored the bypass activity. In addition, DNA polymerase ? misincorporated thymine across the psoralen cross-link and DNA polymerase ? extended these mispaired primer ends, suggesting that DNA polymerase ? may serve as an inserter and DNA polymerase ? may play a role as an extender in the repair of psoralen DNA interstrand cross-links. The results demonstrated here indicate that multiple DNA polymerases could participate in TLS steps in mammalian DNA interstrand cross-link repair.

SUBMITTER: Smith LA

PROVIDER: S-EPMC3516411 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Bypass of a psoralen DNA interstrand cross-link by DNA polymerases β, ι, and κ in vitro.

Smith Leigh A LA Makarova Alena V AV Samson Laura L Thiesen Katherine E KE Dhar Alok A Bessho Tadayoshi T

Biochemistry 20121029 44

Repair of DNA interstrand cross-links in mammalian cells involves several biochemically distinctive processes, including the release of one of the cross-linked strands and translesion DNA synthesis (TLS). In this report, we investigated the in vitro TLS activity of a psoralen DNA interstrand cross-link by three DNA repair polymerases, DNA polymerases β, κ, and ι. DNA polymerase β is capable of bypassing a psoralen cross-link with a low efficiency. Cell extracts prepared from DNA polymerase β kno ...[more]

PMID: 23106263

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Project description:DNA interstrand cross-links (ICLs) are products of chemotherapeutic agents and cellular metabolic processes that block both replication and transcription. If left unrepaired, ICLs are extremely toxic to cells, and ICL repair mechanisms contribute to the survival of certain chemotherapeutic resistance tumors. A critical step in ICL repair involves unhooking the cross-link. In the absence of a homologous donor sequence, the resulting gap can be filled in by a repair synthesis step involving bypass of the cross-link remnant. Here, we examine the effect of cross-link structure on the ability of unhooked DNA substrates to undergo repair synthesis in mammalian whole cell extracts. Using 32P incorporation assays, we found that repair synthesis occurs efficiently past the site of damage when a DNA substrate containing a single N4C-ethyl-N4C cross-link is incubated in HeLa or Chinese hamster ovary cell extracts. This lesion, which can base pair with deoxyguanosine, is readily bypassed by both Escherichia coli DNA polymerase I and T7 DNA polymerase in a primer extension assay. In contrast, bypass was not observed in the primer extension assay or in mammalian cell extracts when DNA substrates containing a N3T-ethyl-N3T or N1I-ethyl-N3T cross-link, whose linkers obstruct the hydrogen bond face of the bases, were used. A modified phosphorothioate sequencing method was used to analyze the ICL repair patches created in the mammalian cell extracts. In the case of the N4C-ethyl-N4C substrate, the repair patch spanned the site of the cross-link, and the lesion was bypassed in an error-free manner. However, although the N3T-ethyl-N3T and N1I-ethyl-N3T substrates were unhooked in the extracts, bypass was not detected. These and our previous results suggest that although the chemical structure of an ICL may not affect initial cross-link unhooking, it can play a significant role in subsequent processing of the cross-link. Understanding how the physical and chemical differences of ICLs affect repair may provide a better understanding of the cytotoxic and mutagenic potential of specific ICLs.

Dataset Information

Bypass of a psoralen DNA interstrand cross-link by DNA polymerases ?, ?, and ? in vitro.

Publications

Bypass of a psoralen DNA interstrand cross-link by DNA polymerases β, ι, and κ in vitro.

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