Slx1-Slx4 Are Subunits of a Structure-specific Endonuclease That Maintains Ribosomal DNA in Fission Yeast

Abstract

In most eukaryotes, genes encoding ribosomal RNAs (rDNA) are clustered in long tandem head-to-tail repeats. Studies of Saccharomyces cerevisiae have indicated that rDNA copy number is maintained through recombination events associated with site-specific blockage of replication forks (RFs). Here, we describe two Schizosaccharomyces pombe proteins, homologs of S. cerevisiae Slx1 and Slx4, as subunits of a novel type of endonuclease that maintains rDNA copy number. The Slx1-Slx4 -dependent endonuclease introduces single-strand cuts in duplex DNA on the 3 side of junctions with single-strand DNA. Deletion of Slx1 or Rqh1 RecQ-like DNA helicase provokes rDNA contraction, whereas simultaneous elimination of Slx1-Slx4 endonuclease and Rqh1 is lethal. Slx1 associates with chromatin at two foci characteristic of the two rDNA repeat loci in S. pombe. We propose a model in which the Slx1-Slx4 complex is involved in the control of the expansion and contraction of the rDNA loci by initiating recombination events at stalled RFs. INTRODUCTIONAccurate duplication of a eukaryotic genome depends on highly proficient DNA replication machinery acting in conjunction with DNA repair and checkpoint signaling pathways Osborn et al., 2002). Repair and checkpoint systems are vital because replisomes stall when they encounter DNA damage, protein complexes, or torsional stress. Arrested replication forks (RFs) are prone to rearrangement or collapse. Studies of bacteria have shown that stalled forks can be rescued through either recombinogenic or nonrecombinogenic pathways (Seigneur et al., 1998;Cox et al., 2000;Seigneur et al., 2000;Cox, 2001;McGlynn and Lloyd, 2002).Recent studies have indicated that eukaryotes rescue stalled forks by mechanisms similar to those proposed to act in prokaryotes (Doe et al., 2000;Cox, 2001). A conserved family of eukaryotic DNA helicases related to Escherichia coli RecQ helicase seems to play a central role in the rescue of stalled forks through a nonrecombinogenic mechanism (Doe et al., 2000;Cox, 2001;Hickson et al., 2001). The crucial role of these RecQ-related helicases in maintenance of genome integrity is underscored by the pleiotropic phenotypes of the human Bloom, Werner, and Rothmund-Thomson syndromes associated with defects in BLM, WRN, and RecQ4 proteins, respectively. These hereditary disorders are characterized by high genomic instability, cancer predisposition and, in the case of Werner syndrome, also premature aging (Shen and Loeb, 2000). In budding yeast, Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe, mutations in the genes encoding the RecQ-related helicases Sgs1 and Rqh1, respectively, are associated with hyper-recombination phenotypes. The sgs1 phenotype is characterized by an increase in intra-and interchromosomal recombination, especially at the tandem repeated ribosomal DNA (rDNA) loci (Watt et al., 1995;Sinclair and Guarente, 1997), whereas rqh1 mutants are unable to segregate their chromosomes properly under conditions that stall replication (Stewart et al., 1997...

Mammalian BTBD12/SLX4 Assembles A Holliday Junction Resolvase and Is Required for DNA Repair

“…Nicked HJs are clipped 2-5 and 3-6 nucleotides 5' of the branch point by S. pombe and S. cerevisiae Mus81 complexes, respectively (Osman et al, 2003;Gaillard et al, 2003). In contrast, S.cerevisiae Slx1p-Slx4p (purified from bacteria) or S. pombe Slx1-Slx4 purified from endogenous sources cleaves migrating HJs in the 3' side of the homologous core Coulon et al, 2004). We found that flap (3'-flap-OE) and replication fork (RF-OE) substrates had a virtually identical pattern of cleavage by both full length SLX4 and SLX4ΔN, and produced major products of 26, 27, and 28 nucleotides in length, reflecting cleavage 3-5 nucleotides 5' to the branch site of these substrates ( Figure 6A, lanes 2, 4, 7, 9).…”

“…Given that yeast Slx1p-Slx4p, like GEN1, cleaves 3' to the junction Coulon et al, 2004) and that SLX1-SLX4 SBD displays the ability to produce nicked duplexes from static HJs ( Figure 5B, lane 13), we hypothesized that the symmetrical cut at nucleotide 32 in X0-BE reflected the activity of SLX1. To address this possibility, we examined the specificity of cleavage by HA-SLX4 SBD complexes, which contain SLX1 but not MUS81-EME1 or ERCC4-ERCC1 (Figure 2, Figure 6E).…”

“…Slx1p contains an N-terminal domain related to the UvrC-intron-endonuclease (URI) class of bacterial endonucleases (known as a GIY domain) and a C-terminal PHD-related domain required for Slx1p activity Coulon et al, 2004). Slx1p binds the Slx4p regulatory subunit, a protein lacking known interaction domains Coulon et al, 2004).…”

“…Slx1p contains an N-terminal domain related to the UvrC-intron-endonuclease (URI) class of bacterial endonucleases (known as a GIY domain) and a C-terminal PHD-related domain required for Slx1p activity Coulon et al, 2004). Slx1p binds the Slx4p regulatory subunit, a protein lacking known interaction domains Coulon et al, 2004). Slx1p-Slx4p displays a preference for 5'-flap structures in vitro and slx4Δ, but not slx1Δ, mutants are highly sensitive to MMS and camptothecin (Flott et al, 2007;Fricke and Brill, 2003;Deng et al, 2005).…”

“…As this is a critical transition, the identity of resolvase enzymes has been a central focus of the field. While MUS81-EME1 (and its yeast ortholog Mus81p-Mms4p) and Slx1p-Slx4p display a preference for flap structures and replication forks, they have also been shown to display activity towards migrating or nicked HJs, albeit in a largely non-symmetrical manner Ciccia et al, 2003;Constantinou et al, 2002;Fricke and Brill, 2003;Osman et al, 2003;Chen et al, 2001;Gaillard et al, 2003;Taylor and McGowan, 2008;Coulon et al, 2004) and Mus81 mutants are defective in resolution of cruciform structures formed by palindromic sequences that mimic a HJ in vivo (Cote and Lewis, 2008). However, MUS81-EME1 and Slx1p-Slx4p prepared in E. coli are largely devoid of activity toward static HJs (Ciccia et al, 2003;Fricke and Brill, 2003) leading to the conclusion that these enzymes alone are not efficient HJ resolvases.…”