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DNA cross-link repair safeguards genomic stability during premeiotic germ cell development.

ABSTRACT: Germline de novo mutations are the basis of evolutionary diversity but also of genetic disease. However, the molecular origin, mechanisms and timing of germline mutagenesis are not fully understood. Here, we define a fundamental role for DNA interstrand cross-link repair in the germline. This repair process is essential for primordial germ cell (PGC) maturation during embryonic development. Inactivation of cross-link repair leads to genetic instability that is restricted to PGCs within the genital ridge during a narrow temporal window. Having successfully activated the PGC transcriptional program, a potent quality control mechanism detects and drives damaged PGCs into apoptosis. Therefore, these findings define a source of DNA damage and the nature of the subsequent DNA repair response in germ cells, which ensures faithful transmission of the genome between generations.

SUBMITTER: Hill RJ

PROVIDER: S-EPMC6675612 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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DNA cross-link repair safeguards genomic stability during premeiotic germ cell development.

Hill Ross J RJ Crossan Gerry P GP

Nature genetics 20190731 8

Germline de novo mutations are the basis of evolutionary diversity but also of genetic disease. However, the molecular origin, mechanisms and timing of germline mutagenesis are not fully understood. Here, we define a fundamental role for DNA interstrand cross-link repair in the germline. This repair process is essential for primordial germ cell (PGC) maturation during embryonic development. Inactivation of cross-link repair leads to genetic instability that is restricted to PGCs within the genit ...[more]

PMID: 31367016

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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

DNA cross-link repair safeguards genomic stability during premeiotic germ cell development.

Publications

DNA cross-link repair safeguards genomic stability during premeiotic germ cell development.

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