A DNA sequence conferring high postmeiotic segregation frequency to heterozygous deletions in Saccharomyces cerevisiae is related to sequences associated with eucaryotic recombination hotspots.

Abstract

The meiotic behavior of two graded series of deletion mutations in the ADE8 gene in Saccharomyces cerevisiae was analyzed to investigate the molecular basis of meiotic recombination. Postmeiotic segregation (PMS) was observed for a subset of the deletion heterozygosities, including deletions of 38 to 93 base pairs. There was no clear relationship between deletion length and PMS frequency. A common sequence characterized the novel joint region in the alleles which displayed PMS. This sequence is related to repeated sequences recently identified in association with recombination hotspots in the human and mouse genomes. We propose that these particular deletion heterozygosities escape heteroduplex DNA repair because of fortulitous homology to a binding site for a protein.Meiotic recombination is the fundamental process which is responsible for the reassortment of genetic information between paired homologous chromosomes in zygotic organisms. The physical recombination event takes place after premeiotic DNA replication but before the first nuclear division (1). At that time, each locus is represented on eight DNA strands, i.e., two double-stranded copies of each homolog. Ordinarily, interaction between homologs results in a detectable exchange of information. This exchange can involve one or two strands of DNA and can be reciprocal or nonreciprocal. A nonreciprocal exchange is termed a gene conversion. Because reciprocal and nonreciprocal exchanges are nonrandomly associated in meiosis, gene conversion can be regarded as a signature of the recombination process (5). We study recombination in Saccharomyces cerevisiae because of the powerful confluence of available molecular and genetic technologies. In particular, tetrad dissection of sporulated, appropriately marked diploid strains, followed by replica plating of the resulting four undisturbed ascosporal colonies, allows us to visualize the ultimate fates of each of the eight DNA strands which were present before the meiotic divisions. In addition, replacement of resident alleles by in vitro-modified sequences is now a routine procedure (2).Postmeiotic segregation (PMS) is detected phenotypically as the sectoring of a single heterozygous marker in a haploid ascosporal colony and represents marker segregation at the first postmeiotic mitosis. Presumably, such segregation results when the spore contains uncorrected heteroduplex DNA which encompasses the heterozygosity. PMS at ADE8 can be visualized as red and white half sectors caused by the interaction of ADE2 and ADE8. PMS in yeast was first observed by Esposito in an ade8-181ADE8 heterozygote (3). In a previous analysis, it was found that PMS frequency can be correlated with the potential heteroduplex DNA mismatch (16) generated by the interacting alleles. Furthermore, among those analyzed, the mutant allele displaying the highest PMS frequency (ade8-18) was determined to harbor a 38-base-pair (bp) deletion. Since previous studies showed that large deletions and frameshift mutations do not display appreciable PMS l...

A poly(dA.dT) tract is a component of the recombination initiation site at the ARG4 locus in Saccharomyces cerevisiae.

“…In contrast to the behavior of A136, deletion A42 does not display significant disparity when heterozygous ( tions, which also display high levels of postmeiotic segregation (34). However A42 does not fit the loose consensus sequence derived from the ADE8 deletions.…”

Translation of the yeast transcriptional activator GCN4 is stimulated by purine limitation: implications for activation of the protein kinase GCN2.

“…This study was undertaken to determine whether the expression of GCN4 increases under conditions in which purines are limiting for the growth of yeast cells. These experiments were motivated by the fact that consensus GCN4 binding sites have been identified in the promoters of the ADE1, ADE2, ADE3, ADE4, ADE5, 7, and ADE8 genes (20,39,41,52,51,58) and a report that GCN4 contributes directly to the transcriptional activation of ADE4 (39). We have shown that starvation for purines in any of several different ways leads to increased GCN4 expression at the translational level by a mechanism that depends on the uORFs in the GCN4 mRNA leader, phosphorylation of eIF-2a by the protein kinase GCN2, and the products of GCN3 and GCNI.…”

“…The ADES, 7 probe was a 2.1-kb HindIII fragment from plasmid pYEADE5,7(5.2R) (20). TheADE8 probe was a 0.3-kb XhoI-NruI fragment from plasmid pR102 (58). A probe corresponding to the 1.4-kb EcoRV-Sall fragment of HIS4 on plasmid p19 (16) was used as a positive control for a gene under GCN4 regulation.…”

“…This carboxyterminal domain is required in vivo for GCN2 positive regulatory function and for the association between GCN2 and ribosomes (45). The sequence similarity between GCN2 and HisRSs, combined with the fact that a reduction in tRNA aminoacylation signals derepression of the general control system, led Wek et al (56) (20), and ADE8 (58). Binding of the GCN4 protein to two of three potential binding sites in the ADE4 promoter was demonstrated by Mosch et al (39), and mutations in these sequences that eliminate binding of GCN4 in vitro decrease ADE4 transcription in vivo.…”

Formation of heteroduplex DNA during mammalian intrachromosomal gene conversion.

“…Since the degree of PMS in fungi is highly dependent on the particular marker involved, specific mismatches in DNA are thought to be corrected to different degrees by the mismatch repair machinery (3,34,35). Recently, Nag et al (24,25) have shown that palindromic insertions appear to elude the mismatch repair machinery and thereby generate high levels of PMS.…”