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The HMCES DNA-protein cross-link functions as an intermediate in DNA interstrand cross-link repair.

ABSTRACT: The 5-hydroxymethylcytosine binding, embryonic stem-cell-specific (HMCES) protein forms a covalent DNA-protein cross-link (DPC) with abasic (AP) sites in single-stranded DNA, and the resulting HMCES-DPC is thought to suppress double-strand break formation in S phase. However, the dynamics of HMCES cross-linking and whether any DNA repair pathways normally include an HMCES-DPC intermediate remain unknown. Here, we use Xenopus egg extracts to show that an HMCES-DPC forms on the AP site generated during replication-coupled DNA interstrand cross-link repair. We show that HMCES cross-links form on DNA after the replicative CDC45-MCM2-7-GINS (CMG) helicase has passed over the AP site, and that HMCES is subsequently removed by the SPRTN protease. The HMCES-DPC suppresses double-strand break formation, slows translesion synthesis past the AP site and introduces a bias for insertion of deoxyguanosine opposite the AP site. These data demonstrate that HMCES-DPCs form as intermediates in replication-coupled repair, and they suggest a general model of how HMCES protects AP sites during DNA replication.

SUBMITTER: Semlow DR

PROVIDER: S-EPMC9949344 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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The HMCES DNA-protein cross-link functions as an intermediate in DNA interstrand cross-link repair.

Semlow Daniel R DR MacKrell Victoria A VA Walter Johannes C JC

Nature structural & molecular biology 20220509 5

The 5-hydroxymethylcytosine binding, embryonic stem-cell-specific (HMCES) protein forms a covalent DNA-protein cross-link (DPC) with abasic (AP) sites in single-stranded DNA, and the resulting HMCES-DPC is thought to suppress double-strand break formation in S phase. However, the dynamics of HMCES cross-linking and whether any DNA repair pathways normally include an HMCES-DPC intermediate remain unknown. Here, we use Xenopus egg extracts to show that an HMCES-DPC forms on the AP site generated d ...[more]

PMID: 35534579

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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

The HMCES DNA-protein cross-link functions as an intermediate in DNA interstrand cross-link repair.

Publications

The HMCES DNA-protein cross-link functions as an intermediate in DNA interstrand cross-link repair.

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