Cytotoxicity of cisplatin and mitomycin C (MMC) is ascribed largely to their ability to generate interstrand crosslinks (ICLs) in DNA, which block the progression of replication forks. The processing of ICLs requires the Fanconi anemia (FA) pathway, excision repair, and translesion DNA synthesis (TLS). It also requires homologous recombination (HR), which repairs double-strand breaks (DSBs) generated by cleavage of the blocked replication forks. Here we describe KIAA1018, an evolutionarily conserved protein that has an N-terminal ubiquitin-binding zinc finger (UBZ) and a C-terminal nuclease domain. KIAA1018 is a 5'-->3' exonuclease and a structure-specific endonuclease that preferentially incises 5' flaps. Like cells from FA patients, human cells depleted of KIAA1018 are sensitized to ICL-inducing agents and display chromosomal instability. The link of KIAA1018 to the FA pathway is further strengthened by its recruitment to DNA damage through interaction of its UBZ domain with monoubiquitylated FANCD2. We therefore propose to name KIAA1018 FANCD2-associated nuclease, FAN1.

Single strand nicks and gaps in DNA have been reported to increase the efficiency of nucleosome loading mediated by chromatin assembly factor 1 (CAF-1). However, on mismatch-containing substrates, these strand discontinuities are utilized by the mismatch repair (MMR) system as loading sites for exonuclease 1, at which degradation of the error-containing strand commences. Because packaging of DNA into chromatin might inhibit MMR, we were interested to learn whether chromatin assembly is differentially regulated on heteroduplex and homoduplex substrates. We now show that the presence of a mismatch in a nicked plasmid substrate delays nucleosome loading in human cell extracts. Our data also suggest that, once the mismatch is removed, repair of the single-stranded gap is accompanied by efficient nucleosome loading. We postulated that the balance between MMR and chromatin assembly might be governed by proliferating cell nuclear antigen (PCNA), the processivity factor of replicative DNA polymerases, which is loaded at DNA termini and which interacts with the MSH6 subunit of the mismatch recognition factor MutSα, as well as with CAF-1. We now show that this regulation might be more complex; MutSα and CAF-1 interact not only with PCNA, but also with each other. In vivo this interaction increases during S-phase and may be controlled by the phosphorylation status of the p150 subunit of CAF-1. MMR has evolved to process mismatches arising during replication or recombination (1, 2). That its malfunction leads to cancer (3) bears witness to the importance of its role in the maintenance of genomic stability. In eukaryotes, mismatch repair (MMR) is initiated by the binding of MutSα, a heterodimer of MSH2 and MSH6, to non-Watson-Crick base pairs in DNA. Exchange of ADP for ATP then converts MutSα into a sliding clamp, which diffuses along the DNA contour, possibly together with a heterodimer of MLH1/PMS2 named MutLα, in search of free DNA termini that serve as initiation sites for exonuclease 1-catalyzed degradation of the error-containing DNA strand (1, 2).In mammalian cells, free termini are generally marked by PCNA, which helps orchestrate replication and repair DNA synthesis through interactions with key players of DNA metabolism, including MSH6 and chromatin assembly factor 1 (CAF-1) (4). The latter complex, composed of p150, p60, and p48 subunits (5), promotes rapid assembly of nucleosomes on newly replicated DNA (6).Nucleosome loading causes supercoiling, which compacts the genome and increases its stability, but which makes DNA less accessible to metabolic processes. Thus, during nucleotide excision repair, efficient processing of UV-induced DNA damage requires chromatin remodeling (7, 8) and CAF-1-mediated chromatin reassembly upon repair completion (9). Whether similar transactions take place during MMR is unknown. As the sliding function of MutSα was reported to be blocked by nucleosomes (10, 11), albeit not in all sequence contexts (12), we argued that mismatches arising during replication would have to be repaired ...

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