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

Abstract

Schizosaccharomyces pombe Rad16 is the ortholog of the XPF structure-specific endonuclease, which is required for nucleotide excision repair and implicated in the single strand annealing mechanism of recombination. We show that Rad16 is important for proper completion of meiosis. In its absence, cells suffer reduced spore viability and abnormal chromosome segregation with evidence for fragmentation. Recombination between homologous chromosomes is increased, while recombination within sister chromatids is reduced, suggesting that Rad16 is not required for typical homolog crossovers but influences the balance of recombination between the homolog and the sister. In vegetative cells, rad16 mutants show evidence for genome instability. Similar phenotypes are associated with mutants affecting Rhp14 XPA but are independent of other nucleotide excision repair proteins such as Rad13 XPG . Thus, the XPF/XPA module of the nucleotide excision repair pathway is incorporated into multiple aspects of genome maintenance even in the absence of external DNA damage.T HE XPF/ERCC1 protein complex is one of several structurespecific endonucleases that function broadly as resolvases in the repair of damaged DNA (Schwartz and Heyer 2011). Rad16/Swi9 is the fission yeast ortholog of endonuclease XPF, which forms a complex with Swi10/Rad23 (ERCC1) (Carr et al. 1994). XPF has a conserved role in nucleotide excision repair (NER) to remove UV-induced lesions in the DNA (Camenisch et al. 2006;Gregg et al. 2011). In humans, mutation of XPF is associated with xeroderma pigmentosum (XP), which causes sun sensitivity and high rates of skin cancer, as well as premature aging and neurological disorders (Camenisch et al. 2006;Gregg et al. 2011). Recent studies suggest XPF mutations are also associated with Fanconi anemia (FA), which requires repair of interstrand crosslinks (ICLs) (Bogliolo et al. 2013;Kashiyama et al. 2013). The canonical model for NER suggests that upon recognition of helix-distorting lesions, the DNA is unwound to form a bubble around the damage. XPA (SpRhp14) loads XPF (SpRad16) and ERCC1 (SpSwi10); XPF cleaves at the 59 end of the bubble while XPG (SpRad13), another endonuclease, cleaves at the 39 end to remove the offending segment (Fagbemi et al. 2011;Schwartz and Heyer 2011). Thus, Schizosaccharomyces pombe rad16 mutants show a decrease in (6-4) photoproduct excision (McCready et al. 1993;Carr et al. 1994).Consistent with these clinical effects, XPF is implicated in multiple mechanisms of genome maintenance (Paques and Haber 1997;Gregg et al. 2011;Schwartz and Heyer 2011). XPF is required for single strand annealing (SSA), a form of double strand break (DSB) repair distinct from typical homologous recombination (HR) (Ma et al. 2003;Kass and Jasin 2010). This occurs when short regions of homology exposed by resection are able to pair, leaving nonhomologous 39 overhangs as substrates for XPF cleavage. In budding yeast, recruitment of ScRad1 XPF and ScRad10 ERCC1 in this pathway depends on interactions with other pro...

A non-genetic meiotic repair program inferred from spore survival values in fission yeast wild isolates: a clue for an epigenetic ratchet-like model of ageing?

“…The results obtained in all the experiments performed with Leupold's derived strains in this study might provide a clear answer to the previously mentioned non-consistent results observed in many studies [66][67][68][69][70][71] . A clear dual behaviour is seen in canonical Leupold's derived fission yeast strains, showing around 80% spore survival, or around 95% spore survival (see Figure 1C), which could clearly explain the previously mentioned non-consistent results, in which in some studies the wild type spore survival is stated as 80% [69][70][71] , while in other studies it is stated as 95% [66][67][68] . Although, this might be simply the case, this study uncovers many more factors affecting the spore survival.…”

“…Although, this might be simply the case, this study uncovers many more factors affecting the spore survival. It cannot be discarded that, any of those other factors, do as well explain the non-consistent results observed in the literature [66][67][68][69][70][71] (see later).…”

“…Only 26 SNPs differences can be detected between L968 and JB32 by full genome sequencing (see Materials and methods and Table S3). This observation, together with the low spore survival values identified in self-crosses, both in the lab 58 and in the literature 78 , and together with the non-consistent results seen in fission yeast papers regarding Leupold's derived strains wild type spore survival [66][67][68][69][70][71] , brought my attention into systematically determine the spore survival values observed in self-crosses from individual Leupold's derived strains. I obtained two biological independent diploid homozygotes of the following strains: L968, L972, L975 and JB32.…”

“…Determination of the spore survival values after a meiotic division in yeast can be precisely measured by means of tetrad analysis (see below). Despite that, the spore survival values of the wild type fission yeast strains (all derived from Leupold's isolates), have been measured, using tetrad analysis, in several previous studies showing no consistent results between them: some studies show wild type survivals close to 95% [66][67][68] , while some others show survivals close to 80% [69][70][71] . Most studies use the determination of wild type spore survival just as a value to compare with the mutant of interest.…”

Suppression of Meiotic Recombination by CENP-B Homologs inSchizosaccharomyces pombe

“…To detect rare meiotic recombinants at a Tf2 element, we constructed two parental strains with each containing either a ura4 + or kan gene cassette positioned either upstream or downstream of an active Tf2-12 element, respectively ( Figure 1A). We performed a random spore analysis (RSA) assay (Mastro and Forsburg 2014) of the meiotic products from a cross of the two parents. Surprisingly, the rate of recombination at Tf2-12 varied from 0.5 to 1%, well-within the range of a typical locus (Young et al 2002).…”