The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires processing of broken ends. For repair to commence, the DSB must first be resected to generate a 3'-single-stranded DNA (ssDNA) overhang, which becomes a substrate for the DNA strand exchange protein, Rad511. Genetic studies have implicated a multitude of proteins in the process, including helicases, nucleases, and topoisomerases2–4. Here we have biochemically reconstituted elements of the resection process and reveal that it requires the nuclease, Dna2, the RecQ-family helicase, Sgs1, and the ssDNA-binding protein, Replication protein-A (RPA). We establish that Dna2, Sgs1, and RPA comprise a minimal protein complex capable of DNA resection in vitro. Sgs1 helicase unwinds the DNA to produce an intermediate that is digested by Dna2, and RPA stimulates DNA unwinding by Sgs1 in a species-specific manner. Interestingly, RPA is also required both to direct Dna2 nucleolytic activity to the 5'-terminated strand of the DNA break and to inhibit 3'→5' degradation by Dna2, actions which generate and protect the 3'-ssDNA overhang, respectively. In addition to this core machinery, we establish that both the topoisomerase 3 (Top3) and Rmi1 complex and the Mre11-Rad50-Xrs2 complex (MRX) play important roles as stimulatory components. Stimulation of end resection by the Top3-Rmi1 heterodimer and the MRX proteins is via complex formation with Sgs15,6 that unexpectedly stimulates DNA unwinding. We suggest that Top3-Rmi1 and MRX are important for recruitment of the Sgs1-Dna2 complex to DSBs. Our experiments provide a mechanistic framework for understanding initial steps of recombinational DNA repair in eukaryotes.

To repair double-strand DNA breaks by homologous recombination, the 5'-terminated DNA strand must first be resected, which generates 3' single-stranded DNA overhangs. Genetic evidence suggests that this process is initiated by the Mre11-Rad50-Xrs2 (MRX) complex. However, its involvement was puzzling, as the complex possesses exonuclease activity with the opposite (3' to 5') polarity from that required for homologous recombination. Consequently, a bidirectional model has been proposed whereby dsDNA is first incised endonucleolytically and MRX then proceeds back to the dsDNA end using its 3' to 5' exonuclease. The endonuclease creates entry sites for Sgs1-Dna2 and/or Exo1, which then carry out long-range resection in the 5' to 3' direction. However, the identity of the endonuclease remained unclear. Using purified Saccharomyces cerevisiae proteins, we show that Sae2 promotes dsDNA-specific endonuclease activity by the Mre11 subunit within the MRX complex. The endonuclease preferentially cleaves the 5'-terminated dsDNA strand, which explains the polarity paradox. The dsDNA end clipping is strongly stimulated by protein blocks at the DNA end, and requires the ATPase activity of Rad50 and physical interactions between MRX and Sae2. Our results suggest that MRX initiates dsDNA break processing by dsDNA endonuclease rather than exonuclease activity, and that Sae2 is the key regulator of this process. These findings demonstrate a probable mechanism for the initiation of dsDNA break processing in both vegetative and meiotic cells.

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.

To repair a DNA double-strand break (DSB) by homologous recombination (HR), the 5'-terminated strand of the DSB must be resected. The human MRE11-RAD50-NBS1 (MRN) and CtIP proteins were implicated in the initiation of DNA end resection, but the underlying mechanism remained undefined. Here, we show that CtIP is a co-factor of the MRE11 endonuclease activity within the MRN complex. This function is absolutely dependent on CtIP phosphorylation that includes the key cyclin-dependent kinase target motif at Thr-847. Unlike in yeast, where the Xrs2/NBS1 subunit is dispensable in vitro, NBS1 is absolutely required in the human system. The MRE11 endonuclease in conjunction with RAD50, NBS1, and phosphorylated CtIP preferentially cleaves 5'-terminated DNA strands near DSBs. Our results define the initial step of HR that is particularly relevant for the processing of DSBs bearing protein blocks.