Rad22^Rad52-dependent Repair of Ribosomal DNA Repeats Cleaved by Slx1-Slx4 Endonuclease

Abstract

Slx1 and Slx4 are subunits of a structure-specific DNA endonuclease that is found in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and other eukaryotic species. It is thought to initiate recombination events or process recombination structures that occur during the replication of the tandem repeats of the ribosomal DNA (rDNA) locus. Here, we present evidence that fission yeast Slx1-Slx4 initiates homologous recombination events in the rDNA repeats that are processed by a mechanism that requires Rad22 (Rad52 homologue) but not Rhp51 (Rad51 homologue). Slx1 is required to generate ϳ50% of the spontaneous Rad22 DNA repair foci that occur in cycling cells. Most of these foci colocalize with the nucleolus, which contains the rDNA repeats. The increased fork pausing at the replication fork barriers in the rDNA repeats in a strain that lacks Rqh1 DNA helicase is further increased by expression of a dominant negative form of Slx1. These data suggest that Slx1-Slx4 cleaves paused replication forks in the rDNA, leading to Rad22-dependent homologous recombination that is used to maintain rDNA copy number. INTRODUCTIONWhile replicating the genome, DNA polymerases will sometimes stall when they encounter DNA lesions, protein complexes bound to DNA, or nucleotide starvation. Stalled forks pose serious threats to genomic integrity because they are prone to collapse or rearrangement (McGlynn and Lloyd, 2002). In contrast, there are many examples of natural replication pause and termination sites in a variety of organisms (Rothstein et al., 2000). These sites are often called replication fork barriers (RFBs). It is somewhat paradoxical that in some cases these "programmed" RFBs are thought to play important roles in maintaining genome integrity.The best-characterized RFBs occur in the ribosomal DNA genes (rDNA), which are present in long tandem repeats at one or a few chromosomal loci in most eukaryotic species, including yeasts and humans (Brewer and Fangman, 1988;Linskens and Huberman, 1988;Little et al., 1993;Gerber et al., 1997;Sanchez et al., 1998). The budding yeast Saccharomyces cerevisiae carries ϳ150 copies of the rDNA genes. The RFB is located in the nontranscribed sequence (NTS) between the 3Ј end of the 35S rRNA gene and the autonomously replicating sequence (ARS). The RFB inhibits replication fork progression in the direction opposite to rDNA transcription. This mechanism helps to prevent collisions between replication forks and the transcription machinery (Takeuchi et al., 2003). The protein fork blocking 1 (Fob1) binds DNA in the RFB region and is essential for RFB activity (Kobayashi and Horiuchi, 1996).The size of the rDNA array in S. cerevisiae is not static. For example, it will contract upon inactivation of RNA polymerase I and expands after RNA polymerase I activity is restored (Kobayashi et al., 1998). Fob1 is required for the mechanisms that control the copy number of rDNA repeats (Kobayashi et al., 2001;Johzuka and Horiuchi, 2002). The current model for the regulation of the rDNA copy number involv...

The Rad52 Homologs Rad22 and Rti1 ofSchizosaccharomyces pombeAre Not Essential for Meiotic Interhomolog Recombination, but Are Required for Meiotic Intrachromosomal Recombination and Mating-Type-Related DNA Repair

“…This is consistent with the interpretation that functions of Rad22 and Rti1 not involved in the repair of Rec12-dependent DNA DSBs do not contribute much to overall spore viability. Such additional functions may involve repair of stalled replication forks in meiotic S phase (Figure 9), consistent with the role of Rad22 in vegetative S phase (Noguchi et al 2004;Coulon et al 2006).…”

Increased Meiotic Crossovers and Reduced Genome Stability in Absence of Schizosaccharomyces pombe Rad16 (XPF)

“…In contrast to budding yeast (Flott et al 2007;Li et al 2008), neither slx4Δ nor saw1Δ is sensitive to UV or MMS in fission yeast ( Figure S3 and (Coulon et al 2006). We observed no synthetic growth defects in double mutants between rad16-249 and either slx4Δ or saw1Δ, and little if any effect on MMS or UV sensitivity compared to rad16-249 alone (Table 1 and Figure S3).…”

“…Instead, Sc Slx4 and Saw1 are proposed to provide an alternative XPF loading complex, and Slx4 with Slx1 forms another structure-specific endonuclease (Lyndaker and Alani 2009;Schwartz and Heyer 2011). In fission yeast, Slx4-Slx1 endonuclease is linked to rDNA maintenance via a Rad51-independent recombination mechanism (Coulon et al 2006), but so far there is no evidence that Slx4 and Saw1 affect Rad16 XPF in S. pombe. Significantly we and others observe no damage sensitivity in slx4Δ or saw1Δ mutants ( Figure S3 and Coulon et al 2006) and no change in nucleolar morphology in rad16-249 (data not shown), suggesting they operate independently.…”

Human SLX4 Is a Holliday Junction Resolvase Subunit that Binds Multiple DNA Repair/Recombination Endonucleases

“…An important source of spontaneous genomic instability comes from the replication of genomic loci rich in repeated sequences such as the rDNA. In yeast, Slx1-Slx4 maintains the stability of the rDNA locus where it is thought to initiate HR by collapsing stalled replication forks Coulon et al, 2006;Kaliraman and Brill, 2002). It is conceivable that SLX1-SLX4 not only initiates HR by collapsing the stalled fork but also resolves HJs generated after fork recapture.…”

“…Slx1 and Slx4, were subsequently shown to be catalytic and regulatory subunits, respectively, of a 5′-flap endonuclease Fricke and Brill, 2003). Studies in S. cerevisiae and S. pombe revealed that Slx1-Slx4 plays a critical role in maintaining the integrity of the ribosomal DNA (rDNA) loci, which are made of tandem rDNA repeats Coulon et al, 2006;Kaliraman and Brill, 2002). Slx1-Slx4 was proposed to initiate DNA recombination by collapsing replication forks stalled at natural replication pause sites.…”