Structural and Mechanistic Analysis of the Slx1-Slx4 Endonuclease

摘要

class="head no_bottom_margin" id="sec1title">IntroductionThe human SLX1-SLX4 structure-selective endonuclease plays a key role in DNA repair, homologous recombination, replication fork restart, and telomere maintenance (). The genes encoding Saccharomyces cerevisiae Slx1 and Slx4 were discovered in a genetic screen for mutations that are synthetic lethal in the absence of the Sgs1 helicase, a protein that is important for genome stability (). Homology searches subsequently identified the SLX1 and SLX4 genes in higher eukaryotes (). Slx1 is an evolutionarily conserved protein that contains an N-terminal GIY-YIG nuclease domain (also called URI domain) and a C-terminal zinc-finger domain. GIY-YIG domains also are present in homing nucleases, the bacterial nucleotide excision-repair nuclease UvrC, and several type II restriction enzymes (). The mechanism of substrate binding and cleavage for GIY-YIG family members has been elucidated by crystallographic studies of protein-DNA complexes obtained for two restrictases, namely R.Eco29kl () and Hpy188I ().The Slx4 subunit of the Slx1-Slx4 nuclease is thought to provide a scaffold that coordinates the actions of a number of proteins involved in DNA processing (). For example, vertebrate SLX4 is a large, multi-domain protein that interacts with several DNA repair proteins (): (1) the N-terminal region of human SLX4 binds the MSH2-MSH3 mismatch-repair complex and XPF-ERCC1 nucleotide excision-repair enzyme; and (2) the C-terminal portion of SLX4 binds the telomeric proteins TRF2 and RAP1, the PLK1 kinase, and the MUS81-EME1 endonuclease. In all organisms studied to date, SLX1 binds to the extreme C-terminal region of SLX4, which contains an evolutionarily conserved helix-turn-helix motif. Interestingly, in vitro studies have shown that SLX4 stimulates the endonuclease activities of SLX1, MUS81-EME1, and XPF-ERCC1 (). The importance of SLX4 is demonstrated by the observation that biallelic mutations in SLX4 (also known as FANCP) are associated with the cancer-prone disorder Fanconi anemia ().Although the amino acid sequence of SLX4 is evolutionarily diverse, the C-terminal region of all SLX4 proteins contains a conserved C-terminal domain (CCD) that underpins the interaction with SLX1 and a DNA-binding SAP domain found in many DNA repair proteins (). In yeast, there are few other discernible domains, whereas SLX4 proteins from higher eukaryotes (e.g., worms, flies, and humans) contain one or two copies of a UBZ family zinc-finger domain known as UBZ4; the MEI9^XPF interaction like region (MLR); and a Broad-complex, Tramtrack, and Bric-a-brac (BTB) domain ().The role of SLX1-SLX4 in DNA repair has been studied extensively (). Although deletion of Slx1 in yeast does not affect the response to DNA damage, it has been shown that Slx1-Slx4 plays a role in maintaining the integrity of ribosomal loci, which contain tandem repeats that frequently lead to replication fork arrest (). It is possible that Slx1-Slx4 is involved in the collapse of stalled forks and the resolution of recombination intermediates, such as Holliday junctions (HJs), after fork recapture (). In human cells, transient depletion of SLX4 leads to an increased sensitivity to alkylating and crosslinking agents, indicating the importance of SLX4 for the repair of DNA inter-strand crosslinks (ICLs) and protein-DNA adducts. Depletion of SLX4 also reduces the efficiency of double-strand break repair and leads to genome instability (href="#bib19" rid="bib19 bib32 bib35 bib41 bib42" class=" bibr popnode">Garner et al., 2013; Muñoz et al., 2009; Sarbajna et al., 2014; Svendsen et al., 2009; Wechsler et al., 2011). Collectively, these observations indicate that SLX1 and/or SLX4 have relatively well-conserved roles in processing DNA intermediates that arise at stalled or collapsed replication forks, particularly when cells are treated with DNA-damaging agents that interfere with normal replication fork progression.Purified S. cerevisiae Slx1-Slx4 cleaves various DNA substrates in vitro, including splayed-arm structures, model replication forks, 5′-flaps, and HJs (href="#bib18" rid="bib18" class=" bibr popnode">Fricke and Brill, 2003). For the 5′-flap substrates, the major cleavage site lies in the 5′ single-stranded arm at the junction between single- and double-stranded DNA. Whereas Slx1 alone possesses weak nuclease activity, the rate is stimulated approximately 500-fold by Slx4. Purified human SLX1-SLX4 exhibits related activities and cleaves 5′-flaps, 3′-flaps, replication forks, and HJs. Of particular interest, SLX1-SLX4 and MUS81-EME1 cooperate during HJ resolution, with SLX1-SLX4 performing the initial nick such that MUS81-EME1 can resolve the nicked HJ without substrate dissociation (href="#bib44" rid="bib44" class=" bibr popnode">Wyatt et al., 2013).Although there is a significant amount of functional information available for Slx1-Slx4 and its associated proteins, structural and mechanistic insights for this enzyme are lacking. Here we report the crystal structure of the Slx1 nuclease, obtained using Candida glabrata Slx1 (Cg-Slx1). The protein forms a compact structure with the GIY-YIG nuclease and RING-finger domains interacting with each other, and the structural arrangement is reinforced by a long α helix. We find that Cg-Slx1 forms a stable homodimer in the absence of Cg-Slx4. Importantly, the crystal structure of Cg-Slx1 in complex with Cg-Slx4^CCD, together with biochemical analyses, demonstrate that Cg-Slx1 homodimerization is mutually exclusive with the formation of a Cg-Slx1-Slx4^CCD heterodimer, revealing a likely regulatory mechanism for Slx1 endonuclease activity.