Rules of Donor Preference in Saccharomyces Mating-Type Gene Switching Revealed by a Competition Assay Involving Two Types of Recombination

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 399-407 ◽  
Author(s):  
Xiaohua Wu ◽  
Cherry Wu ◽  
James E Haber
Keyword(s):  
Mating Type ◽  
Strand Break ◽  
Competition Assay ◽  
A Cells ◽  
Single Strand Annealing ◽  
Type Gene ◽  
Chromosome Iii ◽  
Strand Annealing ◽  
The Right ◽  
Donor Preference

Mating type (MAT) switching in Saccharomyces cerevisiae is initiated by a double-strand break (DSB) created at MAT by HO endonuclease. MAT  a cells activate the entire left arm of chromosome III; thus MAT  a preferentially recombines with the silent donor HML. In contrast, MATα cells inactivate the left arm, including HML, and thus preferentially recombine with HMR, 100 kb to the right of MAT. We present a novel competition assay, in which the DSB at MAT can be repaired either by MAT switching or by single-strand annealing (SSA) between two URA3 genes flanking MAT. With preferred donors, MAT  a or MATα switching occurs 65–70% of the time in competition with SSA. When HML is deleted, 40% of MAT  a cells recombine with the “wrong” donor HMR; however, when HMR is deleted, only 18% of MATα cells recombine with HML. In interchromosomal switching, with donors on chromosome III and MAT on chromosome V, MAT  a retains its strong preference for HML and switching is efficient, when the chromosome III recombination enhancer is present. However, MATα donor preference is lost and interchromosomal switching is very inefficient. These experiments demonstrate the utility of using competition between two outcomes to measure the relative efficiency of recombination.

scholarly journals Mechanism of MAT alpha donor preference during mating-type switching of Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (2) ◽  
pp. 657-668 ◽  
Author(s):  
X Wu ◽  
J K Moore ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Strand Break ◽  
Normal Donor ◽  
Alpha Cells ◽  
Cis Acting ◽  
Mating Type Switching ◽  
Chromosome Iii ◽  
The Right ◽  
Donor Preference

During homothallic switching of the mating-type (MAT) gene in Saccharomyces cerevisiae, a- or alpha-specific sequences are replaced by opposite mating-type sequences copied from one of two silent donor loci, HML alpha or HMRa. The two donors lie at opposite ends of chromosome III, approximately 190 and 90 kb, respectively, from MAT. MAT alpha cells preferentially recombine with HMR, while MATa cells select HML. The mechanisms of donor selection are different for the two mating types. MATa cells, deleted for the preferred HML gene, efficiently use HMR as a donor. However, in MAT alpha cells, HML is not an efficient donor when HMR is deleted; consequently, approximately one-third of HO HML alpha MAT alpha hmr delta cells die because they fail to repair the HO endonuclease-induced double-strand break at MAT. MAT alpha donor preference depends not on the sequence differences between HML and HMR or their surrounding regions but on their chromosomal locations. Cloned HMR donors placed at three other locations to the left of MAT, on either side of the centromere, all fail to act as efficient donors. When the donor is placed 37 kb to the left of MAT, its proximity overcomes normal donor preference, but this position is again inefficiently used when additional DNA is inserted in between the donor and MAT to increase the distance to 62 kb. Donors placed to the right of MAT are efficiently recruited, and in fact a donor situated 16 kb proximal to HMR is used in preference to HMR. The cis-acting chromosomal determinants of MAT alpha preference are not influenced by the chromosomal orientation of MAT or by sequences as far as 6 kb from HMR. These data argue that there is an alpha-specific mechanism to inhibit the use of donors to the left of MAT alpha, causing the cell to recombine most often with donors to the right of MAT alpha.

scholarly journals A Sir2-regulated locus control region in the recombination enhancer of Saccharomyces cerevisiae specifies chromosome III structure

2019 ◽  
Author(s):  
Mingguang Li ◽  
Ryan D. Fine ◽  
Manikarna Dinda ◽  
Stefan Bekiranov ◽  
Jeffrey S. Smith
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Control Region ◽  
Mating Type ◽  
Locus Control Region ◽  
A Cells ◽  
Mating Type Switching ◽  
Chromosome Iii ◽  
The Right ◽  
Donor Preference ◽  
Recombination Enhancer

AbstractThe NAD+-dependent histone deacetylase Sir2 was originally identified in Saccharomyces cerevisiae as a silencing factor for HML and HMR, the heterochromatic cassettes utilized as donor templates during mating-type switching. MATa cells preferentially switch to MATα using HML as the donor, which is driven by an adjacent cis-acting element called the recombination enhancer (RE). In this study we demonstrate that Sir2 and the condensin complex are recruited to the RE exclusively in MATa cells, specifically to the promoter of a small gene within the right half of the RE known as RDT1. We go on to demonstrate that the RDT1 promoter functions as a locus control region (LCR) that regulates both transcription and long-range chromatin interactions. Sir2 represses the transcription of RDT1 until it is redistributed to a dsDNA break at the MAT locus induced by the HO endonuclease during mating-type switching. Condensin is also recruited to the RDT1 promoter and is displaced upon HO induction, but does not significantly repress RDT1 transcription. Instead condensin appears to promote mating-type switching efficiency and donor preference by maintaining proper chromosome III architecture, which is defined by the interaction of HML with the right arm of chromosome III, including MATa and HMR. Remarkably, eliminating Sir2 and condensin recruitment to the RDT1 promoter disrupts this structure and reveals an aberrant interaction between MATa and HMR, consistent with the partially defective donor preference for this mutant. Global condensin subunit depletion also impairs mating type switching efficiency and donor preference, suggesting that modulation of chromosome architecture plays a significant role in controlling mating type switching, thus providing a novel model for dissecting condensin function in vivo.Author summarySir2 is a highly conserved NAD+-dependent protein deacetylase and defining member of the sirtuin protein family. It was identified about 40 years ago in the budding yeast, Saccharomyces cerevisiae, as a gene required for silencing of the cryptic mating-type loci, HML and HMR. These heterochromatic cassettes are utilized as templates for mating-type switching, whereby a programmed DNA double-strand break at the MATa or MATα locus is repaired by gene conversion to the opposite mating type. The preference for switching to the opposite mating type is called donor preference, and in MATa cells, is driven by a cis-acting DNA element called the recombination enhancer (RE). It was believed that the only role for Sir2 in mating-type switching was silencing HML and HMR. However, in this study we show that Sir2 also regulates expression of a small gene (RDT1) in the RE that is activated during mating-type switching. The promoter of this gene is also bound by the condensin complex, and deleting this region of the RE drastically changes chromosome III structure and alters donor preference. The RE therefore appears to function as a complex locus control region (LCR) that links transcriptional control to chromatin architecture, and thus provides a new model for investigating the underlying mechanistic principles of programmed chromosome architectural dynamics.

scholarly journals Mating type–dependent constraints on the mobility of the left arm of yeast chromosome III

2004 ◽  
Vol 164 (3) ◽  
pp. 361-371 ◽  
Author(s):  
Debra A. Bressan ◽  
Julio Vazquez ◽  
James E. Haber
Keyword(s):  
Mating Type ◽  
Three Dimensional ◽  
Cell Mobility ◽  
Yeast Chromosome ◽  
Mating Type Gene ◽  
Type Gene ◽  
Three Dimensional Configuration ◽  
Chromosome Iii ◽  
The Right ◽  
Donor Preference

Mating-type gene (MAT) switching in budding yeast exhibits donor preference. MATa preferentially recombines with HML near the left telomere of chromosome III, whereas MATα prefers HMR near the right telomere. Donor preference is controlled by the recombination enhancer (RE) located proximal to HML. To test if HML is constrained in pairing with MATα, we examined live-cell mobility of LacI-GFP–bound lactose operator (lacO) arrays inserted at different chromosomal sites. Without induction of recombination, lacO sequences adjacent to HML are strongly constrained in both MATα and RE-deleted MATa strains, compared with MATa. In contrast, chromosome movement at HMR or near a telomere of chromosome V is mating-type independent. HML is more constrained in MATa Δre and less constrained in MATa RE+ compared with other sites. Although HML and MATa are not prealigned before inducing recombination, the three-dimensional configuration of MAT, HML, and HMR is mating-type dependent. These data suggest there is constitutive tethering of HML, which is relieved in MATa cells through the action of RE.

scholarly journals Cell Cycle-Dependent Regulation of Saccharomyces cerevisiae Donor Preference during Mating-Type Switching by SBF (Swi4/Swi6) and Fkh1

2006 ◽  
Vol 26 (14) ◽  
pp. 5470-5480 ◽  
Author(s):  
Eric Coïc ◽  
Kaiming Sun ◽  
Cherry Wu ◽  
James E. Haber
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Mating Type ◽  
Epigenetic Modification ◽  
Strand Break ◽  
Mating Type Switching ◽  
Chromosome Iii ◽  
Activation Pathways ◽  
Cycle Dependent ◽  
Donor Preference

ABSTRACT Saccharomyces mating-type switching occurs through a double-strand break-initiated gene conversion event at MAT, using one of two donors located distantly on the same chromosome, HMLα and HMR a. MAT a cells preferentially choose HMLα, a decision that depends on the recombination enhancer (RE) that controls recombination along the left arm of chromosome III. We previously showed that an fhk1Δ mutation reduces HMLα usage in MAT a cells, but not to the level seen when RE is deleted. We now report that donor preference also depends on binding of the Swi4/Swi6 (SBF) transcription factors to an evolutionarily conserved SCB site within RE. As at other SCB-containing promoters, SBF binds to RE in the G1 phase. Surprisingly, Fkh1 binds to RE only in G2, which contrasts with its cell cycle-independent binding to its other target promoters. SBF and Fkh1 define two independent RE activation pathways, as deletion of both Fkh1 and SCB results in nearly complete loss of HML usage in MAT a cells. These transcription factors create an epigenetic modification of RE in a fashion that apparently does not involve transcription. In addition, the putative helicase Chl1, previously involved in donor preference, functions in the SBF pathway.

Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells

1994 ◽  
Vol 14 (1) ◽  
pp. 400-406
Author(s):  
W P Deng ◽  
J A Nickoloff
Keyword(s):  
Dna Replication ◽  
Mismatch Repair ◽  
Mammalian Cells ◽  
Strand Break ◽  
Wild Type ◽  
Heteroduplex Dna ◽  
Frameshift Mutations ◽  
Single Strand Annealing ◽  
Extrachromosomal Recombination ◽  
Strand Annealing

Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication.

scholarly journals RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria

2015 ◽  
Vol 197 (19) ◽  
pp. 3121-3132 ◽  
Author(s):  
Richa Gupta ◽  
Stewart Shuman ◽  
Michael S. Glickman
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Strand Break ◽  
Single Strand ◽  
Central Position ◽  
End Joining ◽  
Dna Double Strand Break ◽  
Single Strand Annealing ◽  
Strand Annealing

ABSTRACTMycobacteria encode three DNA double-strand break repair pathways: (i) RecA-dependent homologous recombination (HR), (ii) Ku-dependent nonhomologous end joining (NHEJ), and (iii) RecBCD-dependent single-strand annealing (SSA). Mycobacterial HR has two presynaptic pathway options that rely on the helicase-nuclease AdnAB and the strand annealing protein RecO, respectively. Ablation ofadnABorrecOindividually causes partial impairment of HR, but loss ofadnABandrecOin combination abolishes HR. RecO, which can accelerate annealing of single-stranded DNAin vitro, also participates in the SSA pathway. The functions of RecF and RecR, which, in other model bacteria, function in concert with RecO as mediators of RecA loading, have not been examined in mycobacteria. Here, we present a genetic analysis ofrecFandrecRin mycobacterial recombination. We find that RecF, like RecO, participates in the AdnAB-independent arm of the HR pathway and in SSA. In contrast, RecR is required for all HR in mycobacteria and for SSA. The essentiality of RecR as an agent of HR is yet another distinctive feature of mycobacterial DNA repair.IMPORTANCEThis study clarifies the molecular requirements for homologous recombination in mycobacteria. Specifically, we demonstrate that RecF and RecR play important roles in both the RecA-dependent homologous recombination and RecA-independent single-strand annealing pathways. Coupled with our previous findings (R. Gupta, M. Ryzhikov, O. Koroleva, M. Unciuleac, S. Shuman, S. Korolev, and M. S. Glickman, Nucleic Acids Res 41:2284–2295, 2013,http://dx.doi.org/10.1093/nar/gks1298), these results revise our view of mycobacterial recombination and place the RecFOR system in a central position in homology-dependent DNA repair.

scholarly journals Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones.

1982 ◽  
Vol 2 (1) ◽  
pp. 11-20 ◽  
Author(s):  
R K Chan ◽  
C A Otte
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Solid Medium ◽  
G1 Arrest ◽  
Alpha Cells ◽  
Opposite Mating Type ◽  
Alpha Factor ◽  
Complementation Groups ◽  
Chromosome Iii ◽  
The Right

Eight independently isolated mutants which are supersensitive (Sst-) to the G1 arrest induced by the tridecapeptide pheromone alpha factor were identified by screening mutagenized Saccharomyces cerevisiae MATa cells on solid medium for increased growth inhibition by alpha factor. These mutants carried lesions in two complementation groups, sst1 and sst2. Mutations at the sst1 locus were mating type specific: MATa sst1 cells were supersensitive to alpha factor, but MAT alpha sst1 cells were not supersensitive to a factor. In contrast, mutations at the sst2 locus conferred supersensitivity to the pheromones of the opposite mating type on both MATa and MAT alpha cells. Even in the absence of added alpha pheromone, about 10% of the cells in exponentially growing cultures of MATa strains carrying any of three different alleles of sst2 (including the ochre mutation sst2-4) had the aberrant morphology ("shmoo" shape) that normally develops only after MATa cells are exposed to alpha factor. This "self-shmooing" phenotype was genetically linked to the sst2 mutations, although the leakiest allele isolated (sst2-3) did not display this characteristic. Normal MATa/MAT alpha diploids do not respond to pheromones; diploids homozygous for an sst2 mutation (MATa/MAT alpha sst2-1/sst2-1) were still insensitive to alpha factor. The sst1 gene was mapped to within 6.9 centimorgans of his6 on chromosome IX. The sst2 gene was unlinked to sst1, was not centromere linked, and was shown to be neither linked to nor centromere distal to MAT on the right arm of chromosome III.

scholarly journals Analysis of BRCA1 Variants in Double-Strand Break Repair by Homologous Recombination and Single-Strand Annealing

2013 ◽  
Vol 34 (3) ◽  
pp. 439-445 ◽  
Author(s):  
William I. Towler ◽  
Jie Zhang ◽  
Derek J. R. Ransburgh ◽  
Amanda E. Toland ◽  
Chikashi Ishioka ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Strand Break ◽  
Double Strand Break ◽  
Single Strand ◽  
Double Strand Break Repair ◽  
Single Strand Annealing ◽  
Break Repair ◽  
Strand Annealing
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