Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (␥-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced ␥-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, ␥-H2AX foci were also induced in Ercc1 MMC-induced ␥-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1؊/؊ and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination.Interstrand cross-links (ICLs) comprise a unique class of DNA lesions that have a potent biological effect. By definition, ICLs involve covalent modification of both strands of DNA. Therefore, these adducts prevent DNA strand separation and block DNA metabolism, such as transcription and replication (31). DNA-damaging agents that cause ICLs are extremely cytotoxic, and their utility as anticancer chemotherapeutics likely stems from their selective toxicity to proliferating cells. ICLs occur via a two-step reaction mechanism in which first a monoadduct involving one strand of DNA is formed (24). Although cross-linking agents induce a variety of DNA adducts, the relative cytotoxicity of each agent correlates with its ability to form ICLs (43, 44).The repair of DNA ICLs presents a unique challenge to cells. Since both strands of DNA are covalently modified, simple excision of the lesion followed by template-driven DNA resynthesis is precluded. In Escherichia coli, two solutions to this problem have been identified (reviewed in reference 19). In both these repair mechanisms, the ICL is excised from one strand. In error-free repair, an undamaged chromosome is then utilized as a template for gap-filling DNA polymerization (55). ICL repair also occurs in recombination-deficient E. coli, likely via translesional DNA polymerization of the second damaged strand (5). Similarly, genetic analysis of Saccharomyces cerevisiae (23) and mammalian DNA repair mutants (reviewed in reference 19) indicates the involvement of proteins from multiple DNA repair pathways in ICL repair: nucleotide excision repair (NER), homologous recombination, and postreplication...

Faithful duplication of the genome requires structure-specific endonucleases such as the RuvABC complex in Escherichia coli. These enzymes help to resolve problems at replication forks that have been disrupted by DNA damage in the template. Much less is known about the identities of these enzymes in mammalian cells. Mus81 is the catalytic component of a eukaryotic structure-specific endonuclease that preferentially cleaves branched DNA substrates reminiscent of replication and recombination intermediates. Here we explore the mechanisms by which Mus81 maintains chromosomal stability. We found that Mus81 is involved in the formation of double-strand DNA breaks in response to the inhibition of replication. Moreover, in the absence of chromosome processing by Mus81, recovery of stalled DNA replication forks is attenuated and chromosomal aberrations arise. We suggest that Mus81 suppresses chromosomal instability by converting potentially detrimental replication-associated DNA structures into intermediates that are more amenable to DNA repair.

Eukaryotic DNA replication terminates when replisomes from adjacent replication origins converge. Termination involves local completion of DNA synthesis, decatenation of daughter molecules, and replisome disassembly. Termination has been difficult to study because termination events are generally asynchronous and sequence non-specific. To overcome these challenges, we paused converging replisomes with a site-specific barrier in Xenopus egg extracts. Upon removal of the barrier, forks underwent synchronous and site-specific termination, allowing mechanistic dissection of this process. We show that DNA synthesis does not slow detectably as forks approach each other and that leading strands pass each other unhindered before undergoing ligation to downstream lagging strands. Dissociation of CMG helicases occurs only after the final ligation step, and is not required for completion of DNA synthesis, strongly suggesting that converging CMGs pass one another and dissociate from double-stranded DNA. This termination mechanism allows rapid completion of DNA synthesis while avoiding premature replisome disassembly

Summary During eukaryotic DNA interstrand cross-link (ICL) repair, cross-links are resolved (“unhooked”) by nucleolytic incisions surrounding the lesion. In vertebrates, ICL repair is triggered when replication forks collide with the lesion, leading to FANCI-FANCD2-dependent unhooking and formation of a double-strand break (DSB) intermediate. Using Xenopus egg extracts, we describe here a replication-coupled ICL repair pathway that does not require incisions or FANCI-FANCD2. Instead, the ICL is unhooked when one of the two N-glycosyl bonds forming the cross-link is cleaved by the DNA glycosylase NEIL3. Cleavage by NEIL3 is the primary unhooking mechanism for psoralen- and abasic site-ICLs. When N-glycosyl bond cleavage is prevented, unhooking occurs via FANCI-FANCD2-dependent incisions. In summary, we identify an incision-independent unhooking mechanism that avoids DSB formation and represents the preferred pathway of ICL repair in a vertebrate cell-free system.