Double strand breaks at the HIS2 recombination hot spot in Saccharomyces cerevisiae

Abstract

Double strand breaks (DSBs) have been found at several meiotic recombination hot spots in Saccharomyces cerevisiae; more global studies have found that they occur at many places along several yeast chromosomes during meiosis. Indeed, the number of breaks found is consistent with the number of recombination events predicted from the genetic map. We have previously demonstrated that the HIS2 gene is a recombination hot spot, exhibiting a high frequency of gene conversion and associated crossing over. This paper shows that DSBs occur in meiosis at a site in the coding region and at a site downstream of the HIS2 gene and that the DSBs are dependent upon genes required for recombination. The frequency of DSBs at HIS2 increases when the gene conversion frequency is increased by alterations in the DNA around HIS2, and vice versa. A deletion that increases both DSBs and conversion can stimulate both when heterozygous; that is, it is semidominant and acts to stimulate DSBs in trans. These data are consistent with the view that homologous chromosomes associate with each other before the formation of the DSBs.The mechanism whereby homologous chromosomes find each other during meiosis and initiate recombination has been the source of considerable speculation and experimentation. Several models have been proposed to account for the genetic and, more recently, molecular data that have been obtained about meiotic recombination. The classical Holliday model (1) suggested that nicks are made in strands of the same polarity in two homologs, after which the broken strands are exchanged from one duplex to the other. The Meselson-Radding model (2) begins recombination with a single strand nick on one homolog, after which one end of the broken strand invades the homologous duplex. More recently, the double strand break (DSB) model for recombination was proposed by Szostak et al. (3); in this scheme, both ends of the broken duplex invade the homolog to initiate recombination. In the latter two models, one can imagine that the mechanism whereby homologs find each other involves recA-like (4) synaptase-assisted searching by the single strands created at the initiating breaks or nicks.Since the publication of the DSB model, experiments have been done that demonstrate that DSBs occur during meiosis in yeast and that they are associated with recombination. These experiments may be sorted into two groups: (i) experiments that examine recombination hot spots both for recombination and DSBs, and (ii) experiments that look at the more global distribution of DSBs along entire chromosomes. Within the first class of experiments, Sun et al. (5) first demonstrated that a meiotic DSB occurs at the ARG4 locus. The location of the DSB was in the DNA sequences that had been shown to be necessary (6) for conversion at ARG4. Furthermore, the time at which the breaks appeared was consistent with the time at which commitment to recombination occurred. In these early experiments, the DSBs were difficult to detect, in part because they were transien...

Clustering of meiotic double-strand breaks on yeast chromosome III

“…At the ARG4 locus, however, DSB formation is inhibited by overlapping transcription (29). The cases of DSBs occurring in the coding regions of GLK1 and HIS2 genes (9,20) show that DSB inhibition by transcription through a gene can be overcome. Still to be elucidated is whether the formation of DSBs within coding regions is controlled by internal specific sequences or by distant cis-acting elements.…”

“…(i) Most DSB sites are found located in intergenic regions containing transcriptional promoters (reviewed in ref. 1) but the occurrence of DSBs at other locations has been reported, including within the coding sequence of the HIS2 gene (9) and at uncharacterized positions in several artificial constructs (10). (ii) The occurrence of DSBs is controlled by determinants of chromatin structure, as they occur in regions that are hypersensitive to DNaseI and micrococcal nuclease (8,(10)(11)(12)(13).…”

Characterization of rec15, an early meiotic recombination gene in Schizosaccharomyces pombe

“…Cervantes et al (2000) showed that double-strand break formation is strongly reduced in a rec7 mutant. This is also the case in mutants of REC114, the homologous early recombination gene of S. cerevisiae (Pittman et al 1993;Bullard et al 1996). S. cerevisiae rec114 mutants and other early recombination mutants undergo meiosis I earlier than wild type.…”

“…rec102, rec104, and rec114 (Bullard et al 1996); mre2 rec7 gene disruption: Two gene disruption mutations were constructed (Figure 1) 1977). Individual chromosomes can be traced used to transform strain SA2 to Ura ϩ by the lithium acetate through meiosis by the use of appropriate markers and method (Ito et al 1983 menting ade6-M210/ade6-M216 colonies are white.…”

Mek1 Suppression of Meiotic Double-Strand Break Repair Is Specific to Sister Chromatids, Chromosome Autonomous and Independent of Rec8 Cohesin Complexes

“…Chromosome III hotspot probes were derived from pME1210 (YCR048w) (Woltering et al 2000) and pNH90 (HIS4/ LEU2) (Hunter and Kleckner 2001). The chromosome VI hotspot (HIS2) was detected using pH 21 (Bullard et al 1996) (provided by Bob Malone) and the chromosome VIII hotspot (ARG4) used pMJ77 (provided by Michael Lichten). The mek1-as allele in pJR2 was constructed by subcloning a 3.2-kb EcoRI/ SalI fragment from pB131-Q241G (Niu et al 2009) into EcoRI/ SalI-digested Ylp5 (Parent et al 1985).…”