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...

Pluripotent stem cells exist in naive and primed states, epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs; ref. 1). In the naive state of ESCs, the genome has an unusual open conformation and possesses a minimum of repressive epigenetic marks2. In contrast, EpiSCs have activated the epigenetic machinery that supports differentiation towards the embryonic cell types3–6. The transition from naive to primed pluripotency therefore represents a pivotal event in cellular differentiation. But the signals that control this fundamental differentiation step remain unclear. We show here that paracrine and autocrine Wnt signals are essential self-renewal factors for ESCs, and are required to inhibit their differentiation into EpiSCs. Moreover, we find that Wnt proteins in combination with the cytokine LIF are sufficient to support ESC self-renewal in the absence of any undefined factors, and support the derivation of new ESC lines, including ones from non-permissive mouse strains. Our results not only demonstrate that Wnt signals regulate the naive-to-primed pluripotency transition, but also identify Wnt as an essential and limiting ESC self-renewal factor.

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.

Repair of interstrand crosslinks (ICLs) requires multiplestrand incisions to separate the two covalently attached strands of DNA. It is unclear how these incisions are generated. DNA double-strand breaks (DSBs) have been identified as intermediates in ICL repair, but enzymes responsible for producing these intermediates are unknown. Here we show that Mus81, a component of the Mus81-Eme1 structure-specific endonuclease, is involved in generating the ICL-induced DSBs in mouse embryonic stem (ES) cells in S phase. Given the DNA junction cleavage specificity of Mus81-Eme1 in vitro, DNA damage-stalled replication forks are suitable in vivo substrates. Interestingly, generation of DSBs from replication forks stalled due to DNA damage that affects only one of the two DNA strands did not require Mus81. Furthermore, in addition to a physical interaction between Mus81 and the homologous recombination protein Rad54, we show that Mus81 À/À Rad54 À/À ES cells were as hypersensitive to ICL agents as Mus81 À/À cells. We propose that Mus81-Eme1-and Rad54-mediated homologous recombination are involved in the same DNA replication-dependent ICL repair pathway.