Abrogation of the Chk1-Pds1 Checkpoint Leads to Tolerance of Persistent Single-Strand Breaks in Saccharomyces cerevisiae

Abstract

In budding yeast, Apn1, Apn2, Tpp1, and Rad1/Rad10 are important enzymes in the removal of spontaneous DNA lesions. apn1 apn2 rad1 yeast are inviable due to accumulation of abasic sites and strand breaks with 3Ј blocking lesions. We found that tpp1 apn1 rad1 yeast exhibited slow growth but frequently gave rise to spontaneous slow growth suppressors that segregated as single-gene mutations. Using a candidate gene approach, we identified several tpp1 apn1 rad1 suppressors. Deleting uracil glycosylase suppressed both tpp1 apn1 rad1 and apn1 apn2 rad1 growth defects by reducing the abasic site burden. Mutants affecting the Chk1-Pds1 metaphase-anaphase checkpoint only suppressed tpp1 apn1 rad1 slow growth. In contrast, most S-phase checkpoint mutants were synthetically lethal in a tpp1 apn1 rad1 background. Epistasis analyses showed an additive effect between chk1 and ung1, indicating different mechanisms of suppression. Loss of Chk1 partially restored cell-growth parameters in tpp1 apn1 rad1 yeast, but at the same time exacerbated chromosome instability. We propose a model in which recombinational repair during S phase coupled with failure of the metaphase-anaphase checkpoint allows for tolerance of persistent single-strand breaks at the expense of genome stability. ous lesion leading to toxicity in these strains.gene replacement was also confirmed by the loss of product in PCR using primers in the coding region of the targeted gene.We have found that a haploid strain lacking Apn1, et al. 1994Sanchez et al. 1999). There is the samples were then measured at 15-to 30-min intervals by using a GENESYS20 spectrophotometer for at least two considerable overlap in the proteins required for these from at least six different isolates were analyzed. As with theIn this study we found that tpp1 apn1 rad1 mutant tetrad scores, this was done prior to genotyping, so that all yeast spontaneously gave rise to single-gene slow-growth data were recorded blind to the strain genotype.suppressors at a high frequency. Using a candidate gene Plasmid loss assay: Plasmid loss rates were determined using the method described by Huang and Koshland (2003).approach, we observed that disruption of the M-A checkBriefly, YW1495, a diploid strain carrying the pRS412 plasmid point conferred suppression of tpp1 apn1 rad1 slow with the CEN/ARS and the ADE2 marker gene, was dissected growth. In contrast, deletion of S-phase checkpoint on selective media plates to get the strains with appropriate genes either had no effect or conferred synthetic lethalgenotypes and containing the plasmid. Either a 6-day tetrad ity in a tpp1 apn1 rad1 mutant background. The implicacolony or a wild-type strain (YW465) carrying the plasmid was used to inoculate the initial culture and allowed to grow in tions of these findings are discussed.YPAD for approximately six to seven doublings. In the case of slow-growing strains, three or more colonies were pooled to form the initial inoculum. The plasmid loss rate was calculated MATERIALS AND METHODS using the formula: l...

TheSaccharomyces cerevisiaecheckpoint genes RAD9, CHK1 and PDS1 are required for elevated homologous recombination in a mec1 (ATR) hypomorphic mutant

“…chk1 mutants tolerate persistent single-strand breaks in DNA. 33 Our results extend these studies by suggesting that CHK1 is required for homologous recombination triggered by S phase checkpoint defects. Since Pds1 activation in G 2 is CHK1-dependent and mec1-21 pds1 mutants are defective in radiation-associated G 2 arrest, we do not fully understand why mec1-21 pds1 mutants still exhibit a modest hyper-recombination phenotype.…”

DNA repair defects sensitize cells to anticodon nuclease yeast killer toxins

“…A slight suppression of apn1 apn2 associated MMS sensitivity could also be observed upon concomitant removal of Ung1 (Fig. 3a), which blocks AP site formation due to the excision of naturally occurring uracil residues in DNA (Karumbati and Wilson 2005;Dornfeld and Johnson 2005). A faint suppression upon Ung1 removal in this Fig.…”

Host Factors That Control Long Terminal Repeat Retrotransposons in Saccharomyces cerevisiae : Implications for Regulation of Mammalian Retroviruses

“…Moreover, Ty1 cDNA levels were decreased in 44 of the 99 hypotransposition mutants found by Griffith et al We examined the genetic interaction data for these 44 genes in the Saccharomyces Genome Database (http://www.yeastgenome .org) to determine whether they would interact in common with genes in particular cellular pathways or processes. While the 44 genes are involved in diverse cellular processes, 33 of them show synthetic genetic interactions with genes that are components of DNA damage-signaling networks or that have roles in DNA replication or repair (2,62,73,81,103). Perhaps a central pathway that is influenced by many genes, such as a DNA damage-signaling pathway, is responsible for the common phenotype of many of these diverse genes.…”