Summary Repair of DNA interstrand crosslinks requires action of multiple DNA repair pathways, including homologous recombination. Here, we report a de novo heterozygous T131P mutation in RAD51/FANCR, the key recombinase essential for homologous recombination, in a patient with Fanconi anemia-like phenotype. In vitro, RAD51-T131P displays DNA-independent ATPase activity, no DNA pairing capacity and a co-dominant negative effect on RAD51 recombinase function. However, the patient cells are homologous recombination proficient due to the low ratio of mutant to wildtype RAD51 in cells. Instead, patient cells are sensitive to crosslinking agents and display hyperphosphorylation of Replication Protein A due to increased activity of DNA2 and WRN at the DNA interstrand crosslinks. Thus, proper RAD51 function is important during DNA interstrand crosslink repair outside of homologous recombination. Our study provides a molecular basis for how RAD51 and its associated factors may operate in a homologous recombination-independent manner to maintain genomic integrity.

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 59-39 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 replicationassociated 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.

Fanconi anemia is a genetic disorder resulting from biallelic mutations in one of the 17 FANC genes. It is characterized by congenital abnormalities, bone marrow failure, and cancer predisposition. The underlying cause is genomic instability resulting from the deficiency in replication-dependent DNA interstrand crosslink repair pathway commonly referred to as the Fanconi anemia-BRCA pathway. This SnapShot presents the key factors involved.

Interstrand cross-links (ICLs) prevent DNA strand separation and, therefore, transcription and replication, making them extremely cytotoxic. The precise mechanism by which ICLs are removed from mammalian genomes largely remains elusive. Genetic evidence implicates ATR, the Fanconi anemia proteins, proteins required for homologous recombination, translesion synthesis, and at least two endonucleases, MUS81-EME1 and XPF-ERCC1. ICLs cause replication-dependent DNA double-strand breaks (DSBs), and MUS81-EME1 facilitates DSB formation. The subsequent repair of these DSBs occurs via homologous recombination after the ICL is unhooked by XPF-ERCC1. Here, we examined the effect of the loss of either nuclease on FANCD2 monoubiquitination to determine if the nucleolytic processing of ICLs is required for the activation of the Fanconi anemia pathway. FANCD2 was monoubiquitinated in Mus81 ؊/؊ , Ercc1 ؊/؊ , and XPF-deficient human, mouse, and hamster cells exposed to cross-linking agents. However, the monoubiquitinated form of FANCD2 persisted longer in XPF-ERCC1-deficient cells than in wild-type cells. Moreover, the levels of chromatin-bound FANCD2 were dramatically reduced and the number of ICL-induced FANCD2 foci significantly lower in XPF-ERCC1-deficient cells. These data demonstrate that the unhooking of an ICL by XPF-ERCC1 is necessary for the stable localization of FANCD2 to the chromatin and subsequent homologous recombination-mediated DSB repair.The XPF-ERCC1 heterodimer is a structure-specific endonuclease that incises double-strand DNA immediately adjacent to a 3Ј-single-stranded region, removing 3Ј overhangs or opening bubbles (12, 69). ERCC1 is required for DNA binding (74), and XPF harbors the catalytic domain (17). XPF-ERCC1 makes the incision 5Ј to the lesion during nucleotide excision repair (NER), the pathway responsible for removing helixdistorting DNA lesions (69). Defects in NER cause xeroderma pigmentosum (XP), a syndrome characterized by photosensitivity and a dramatically increased risk of skin cancers due to failure to repair UV photolesions. Cells from all XP complementation groups (XP-A to XP-G) and the recently reported ERCC1-deficient patient (33) are hypersensitive to UV irradiation. However, cells deficient in XPF-ERCC1 differ from other XP cells in that they also are exquisitely sensitive to chemicals that induce DNA interstrand cross-links (ICLs) (13,28,54). ICLs are extremely cytotoxic lesions formed when bifunctional agents covalently link both strands of DNA, preventing strand separation, which is necessary for replication or transcription (46). Cross-linking agents such as nitrogen mustards (HN2) (37) and mitomycin C (MMC) (31) produce a mixture of monoadducts and ICLs. However, cytotoxicity correlates with the number of ICLs formed rather than monoadducts (60, 62).ICLs present a unique challenge to cells, in that they affect both strands of DNA and therefore cannot be repaired by a simple excision and resynthesis mechanism. The mechanism of ICL repair in Escherichia coli is well characteri...