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One of the major DNA interstrand cross-link (ICL) repair pathways in mammalian cells is coupled to replication, but the mechanistic roles of the critical factors involved remain largely elusive. Here, we show that purified human SNM1A (hSNM1A), which exhibits a 5'-3' exonuclease activity, can load from a single DNA nick and digest past an ICL on its substrate strand. hSNM1A-depleted cells are ICL-sensitive and accumulate replication-associated DNA double-strand breaks (DSBs), akin to ERCC1-depleted cells. These DSBs are Mus81-induced, indicating that replication fork cleavage by Mus81 results from the failure of the hSNM1A- and XPF-ERCC1-dependent ICL repair pathway. Our results reveal how collaboration between hSNM1A and XPF-ERCC1 is necessary to initiate ICL repair in replicating human cells.

Xeroderma pigmentosum (XP) is caused by defects in the nucleotide excision repair (NER) pathway. NER removes helix-distorting DNA lesions, such as UV-induced photodimers, from the genome. Patients suffering from XP exhibit exquisite sun sensitivity, high incidence of skin cancer, and in some cases neurodegeneration. The severity of XP varies tremendously depending upon which NER gene is mutated and how severely the mutation affects DNA repair capacity. XPF-ERCC1 is a structure-specific endonuclease essential for incising the damaged strand of DNA in NER. Missense mutations in XPF can result not only in XP, but also XPF-ERCC1 (XFE) progeroid syndrome, a disease of accelerated aging. In an attempt to determine how mutations in XPF can lead to such diverse symptoms, the effects of a progeria-causing mutation (XPF(R153P)) were compared to an XP-causing mutation (XPF(R799W)) in vitro and in vivo. Recombinant XPF harboring either mutation was purified in a complex with ERCC1 and tested for its ability to incise a stem-loop structure in vitro. Both mutant complexes nicked the substrate indicating that neither mutation obviates catalytic activity of the nuclease. Surprisingly, differential immunostaining and fractionation of cells from an XFE progeroid patient revealed that XPF-ERCC1 is abundant in the cytoplasm. This was confirmed by fluorescent detection of XPF(R153P)-YFP expressed in Xpf mutant cells. In addition, microinjection of XPF(R153P)-ERCC1 into the nucleus of XPF-deficient human cells restored nucleotide excision repair of UV-induced DNA damage. Intriguingly, in all XPF mutant cell lines examined, XPF-ERCC1 was detected in the cytoplasm of a fraction of cells. This demonstrates that at least part of the DNA repair defect and symptoms associated with mutations in XPF are due to mislocalization of XPF-ERCC1 into the cytoplasm of cells, likely due to protein misfolding. Analysis of these patient cells therefore reveals a novel mechanism to potentially regulate a cell's capacity for DNA repair: by manipulating nuclear localization of XPF-ERCC1., Competing Interests: The authors have declared that no competing interests exist.

XPF-ERCC1 is a structure-specific endonuclease involved in nucleotide excision repair, interstrand crosslink repair and homologous recombination. So far, it has not been shown experimentally which subunit of the heterodimer harbors the nuclease activity and which amino acids contribute to catalysis. We used an affinity cleavage assay and located the active site to amino acids 670-740 of XPF. Point mutations generated in this region were analyzed for their role in nuclease activity, metal coordination and DNA binding. Several acidic and basic residues turned out to be required for nuclease activity, but not DNA binding. The separation of substrate binding and catalysis by XPF-ERCC1 will be invaluable in studying the role of this protein in various DNA repair processes. Alignment of the active site region of XPF with proteins belonging to the Mus81 family and a putative archaeal RNA helicase family reveals that seven of the residues of XPF involved in nuclease activity are absolutely conserved in the three protein families, indicating that they share a common nuclease motif.