Heteroduplex formation and mismatch repair of the "stuck" mutation during mating-type switching in Saccharomyces cerevisiae (original) (raw)
Related papers
Genetics
Homothallic strains of Saccharomyces cerevisiae are able to switch from one mating-type to the other as frequently as every cell division. We have identified a cis-dominant mutation of the MATa locus, designated MATa-inc, that can be converted to M A T a at only about 5% of the normal efficiency. In homothallic MATa-inc/mata* diploids, the MATa-inc locus switched to MATa in only one of 30 cases, while the rnata* locus switched to MATa in all 30 cases. The MATa-inc mutation can be "healed" by a series of switches, first to MATa and then to a normal allele of MATa. These data are consistent with the "cassette" model of HICKS, STRATHERN and HERSKOWITZ (1977), in which mating conversions involve the transposition of wild-type copies of a or a information from silent genes elsewhere in the genome. The MATa-inc mutation appears to alter a DNA sequence necessary for the replacement of MATa by MATa. The MATa-inc mutation has no other effect on MATa functions. In heterothallic backgrounds, the mutation has no effect on the sensitivity to a-factor, synthesis of a-factor, expression of barrier phenotype or ability to mate or sporulate.-The MATa-inc allele does, however, exhibit one pleiotropic effect. About 1% o€ homothallic MATa-inc cells become completely unable to switch mating type because of mutations at HMa, the locus proposed to carry the silent copy of a information.---In addition, we have isolated a less efficient allele o€ the HO gene.
Genetics
HO endonuclease-induced double-strand breaks (DSBs) within a direct duplication of Eschm'chia coli lacZ genes are repaired either by gene conversion or by single-strand annealing (SSA), with >80% being SSA. Previously it was demonstrated that the RAD52 gene is required for DSB-induced SSA. In the present study, the effects of other genes belonging to the RAD52 epistasis group were analyzed. We show that RAD51, RAD54, RAD55, and RAD57 genes are not required for SSA irrespective of whether recombination occurred in plasmid or chromosomal DNA. In both plasmid and chromosomal constructs with homologous sequences in direct orientation, the proportion of SSA events over gene conversion was significantly elevated in the mutant strains. However, gene conversion was not affected when the two laczsequences were in inverted orientation. These results suggest that there is a competition between SSA and gene conversion processes that favors SSA in the absence of RAD51, RAD54, RAD55 and RAD57. Mutations in RAD50 and XRS2 genes do not prevent the completion, but markedly retard the kinetics, of DSB repair by both mechanisms in the lacZ direct repeat plasmid, a result resembling the effects of these genes during mating-type ( M A 7 ) switching.
Genetics, 1996
HO endonuclease-induced double-strand breaks (DSBs) within a direct duplication of Eschm'chia coli lacZ genes are repaired either by gene conversion or by single-strand annealing (SSA), with >80% being SSA. Previously it was demonstrated that the RAD52 gene is required for DSB-induced SSA. In the present study, the effects of other genes belonging to the RAD52 epistasis group were analyzed. We show that RAD51, RAD54, RAD55, and RAD57 genes are not required for SSA irrespective of whether recombination occurred in plasmid or chromosomal DNA. In both plasmid and chromosomal constructs with homologous sequences in direct orientation, the proportion of SSA events over gene conversion was significantly elevated in the mutant strains. However, gene conversion was not affected when the two laczsequences were in inverted orientation. These results suggest that there is a competition between SSA and gene conversion processes that favors SSA in the absence of RAD51, RAD54, RAD55 and RAD57. Mutations in RAD50 and XRS2 genes do not prevent the completion, but markedly retard the kinetics, of DSB repair by both mechanisms in the lacZ direct repeat plasmid, a result resembling the effects of these genes during mating-type ( M A 7 ) switching.
Molecular and cellular biology, 1985
Homothallic switching of the mating type genes of Saccharomyces cerevisiae occurs by a gene conversion event, replacing sequences at the expressed MAT locus with a DNA segment copied from one of two unexpressed loci, HML or HMR. The transposed Ya or Y alpha sequences are flanked by homologous regions that are believed to be essential for switching. We examined the transposition of a mating type gene (hmr alpha 1-delta 6) which contains a 150-base-pair deletion spanning the site where the HO endonuclease generates a double-stranded break in MAT that initiates the gene conversion event. Despite the fact that the ends of the cut MAT region no longer share homology with the donor hmr alpha 1-delta 6, switching of MATa or MAT alpha to mat alpha 1-delta 6 was efficient. However, there was a marked increase in the number of aberrant events, especially the formation of haploid-inviable fusions between MAT and the hmr alpha 1-delta 6 donor locus.
Genetics
We have investigated HO endonuclease-induced double-strand break (DSB) recombination and repair in a LACZ duplication plasmid in yeast. A 1 17-bp MATa fragment, embedded in one copy of LACZ, served as a site for initiation of a DSB when HO endonuclease was expressed. The DSB could be repaired using wild-type sequences located on a second, promoterless, copy of LACZ on the same plasmid. In contrast to normal mating-type switching, crossing-over associated with gene conversion occurred at least 50% of the time. The proportion of conversion events accompanied by exchange was greater when the two copies of LACZ were in direct orientation (80%), than when inverted (50%). In addition, the fraction of plasmids lost was significantly greater in the inverted orientation. The kinetics of appearance of intermediates and final products were also monitored. The repair of the DSB is slow, requiring at least an hour from the detection of the HO-cut fragments to completion of repair. Surprisingly, the appearance of the two reciprocal products of crossing over did not occur with the same kinetics. For example, when the two LACZ sequences were in the direct orientation, the HO-induced formation of a large circular deletion product was not accompanied by the appearance of a small circular reciprocal product. We suggest that these differences may reflect two kinetically separable processes, one involving only one cut end and the other resulting from the concerted participation of both ends of the DSB.
Proceedings of the National Academy of Sciences, 1983
Several cis-acting mutations that prevent homothallic mating type conversions in Saccharomyces cerevisie have been examined. Deletions within the mating type (MAT) locus were obtained by selecting for survivors among homothallic MATa cells carrying the rad52 mutation. The survivors were unable to switch mating type, even in RADW derivatives. The deletions varied in size from fewer than 50 to more than 750 base pairs. All of the deletions removed a Hha I site at the border between the a-specific sequences (Ya) and the adjacent Z region. We also examined several spontaneous inc mutations that prevent MAT switching. Two of these mutations were cloned in recombinant DNA plasmids and their sequences were determined. The MATa-inc 3-7 mutation proved to have an altered Hha I site at the Ya/Z border, by virtue of a single base pair substitution G'C --A-T in the second base pair of the Z region (Z2). Restriction fragment analysis showed that two other independently isolated strains with MATainc mutations had altered the same Hha I site. The MATa-inc 4-28 mutation contains a single base pair substitution COG -+ TA at position Z6. A base pair difference at position Zn1 in two MATa strains does not affect MATa conversions. We conclude that the region near the Y/Z border is essential for the efficient switching of MAT alleles and constitutes an enzyme recognition site for a specific nucleolytic cleavage of MAT DNA.
Genes & Development, 1995
During mating-type gene switching in Saccharomyces cerevisiae, DNA at the MAT locus is replaced by sequences copied from one of two unexpressed donor loci, HML or HAIR, located near the two ends of the same chromosome and I>90 kb from MAT. MATa cells recombine nearly 90% of the time with HML, whereas MAT, v cells select HAIR. MATa donor preference was examined by deleting HML and inserting a donor at other chromosome III locations. MATa activated a large (~>40 kb) region near the left end of chromosome III, such that a donor placed at several sites within this domain was strongly preferred over HAIR. When inserted outside of this domain, the donor was used equally with HAIR. MATa donor preference for HML was abolished by the expression of the negative regulator, MATer2; however, HML regained its preferred status when the donor was unsilenced. Mating-type-dependent activation of the left end of the chromosome is also observed for other types of recombination that do not involve MAT switching. Spontaneous recombination between two leu2 alleles is 20-30 times higher in MATa than in MATer when one of the leu2 alleles is inserted in place of HML. Transcription in this donor activation region is not affected by mating type. We conclude that MATa donor preference involves a mating-type-regulated change in the accessibility of a large chromosomal domain for recombination.
Mating-Type Gene Switching in Saccharomyces Cerevisiae
Annual Review of Genetics, 1998
Saccharomyces cerevisiae can change its mating type as often as every generation by a highly choreographed, site-specific recombination event that replaces one MAT allele with different DNA sequences encoding the opposite allele. The study of this process has yielded important insights into the control of cell lineage, the silencing of gene expression, and the formation of heterochromatin, as well as the molecular events of double-strand break-induced recombination. In addition, MAT switching provides a remarkable example of a small locus control region-the Recombination Enhancer-that controls recombination along an entire chromosome arm.
Mating-Type Genes and MAT Switching in Saccharomyces cerevisiae
Genetics, 2012
Mating type in Saccharomyces cerevisiae is determined by two nonhomologous alleles, MATa and MATa. These sequences encode regulators of the two different haploid mating types and of the diploids formed by their conjugation. Analysis of the MATa1, MATa1, and MATa2 alleles provided one of the earliest models of cell-type specification by transcriptional activators and repressors. Remarkably, homothallic yeast cells can switch their mating type as often as every generation by a highly choreographed, site-specific homologous recombination event that replaces one MAT allele with different DNA sequences encoding the opposite MAT allele. This replacement process involves the participation of two intact but unexpressed copies of mating-type information at the heterochromatic loci, HMLa and HMRa, which are located at opposite ends of the same chromosome-encoding MAT. The study of MAT switching has yielded important insights into the control of cell lineage, the silencing of gene expression, the formation of heterochromatin, and the regulation of accessibility of the donor sequences. Real-time analysis of MAT switching has provided the most detailed description of the molecular events that occur during the homologous recombinational repair of a programmed double-strand chromosome break.