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Chapter 9 : Decisions, Decisions: Donor Preference during Budding Yeast Mating-Type Switching
Category: Fungi and Fungal Pathogenesis
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Mating-type (MAT) switching in the budding yeast Saccharomyces cerevisiae has been particularly well studied, both in terms of the homologous recombination mechanism that leads to the switch itself and the remarkable donor preference system that ensures efficient exchange of mating types. For example, mutations that prevent acetylation of four lysines in histone H4 lead to the complete unsilencing of HMLa but have much less effect on the silencing of HMRa. MAT switching occurs by a mechanism of DSB repair known as synthesis-dependent strand annealing (SDSA). There are a number of distinct, slow steps in MAT switching that have been identified by a combination of Southern blot and PCR analyses, as well as by chromatin immunoprecipitation techniques, that monitor the recruitment of recombination proteins to the sequences undergoing recombination/repair. Analysis of various temperature-sensitive mutations in essential DNA replication genes has shown that the short patch of new DNA synthesis in MAT switching does not require the Mcm helicase proteins or Pol-primase or the Cdc7 kinase. The binding of both Fkh1 and Swi4/6 to recombination enhancer (RE) has been evaluated by chromatin immunoprecipitation at different times in the cell cycle. RE represents one of the most fascinating and best-defined cis-acting loci that control aspects of chromosome positioning, folding, or tethering. The availability of new cytological resources as well as a wide variety of molecular and genetic tools opens the way to a detailed understanding of the ways chromosomes are arranged and constrained in the nucleus.
Mating-type (MAT) gene switching in S. cerevisiae. (A) HO endonuclease cleavage of MAT a promotes homologous recombination with one of two donor loci, in this case HMLα, to repair the locus and to replace the Ya sequences with Y sequences. Ya and Y encode regulators of a-, α-, and haploid-specific genes to establish the cell’s mating type. MAT shares homology with both HML and HMR in the X and Z1 regions and shares additional homology with HML in W and Z2. Both HML and HMR are maintained in an unexpressed and heterochromatic state (shown as hatched lines) by Sir2-mediated gene silencing that is organized through the E and I silencer sequences. The preferential use of HML by MAT a cells and the selection of HMR by MATα cells is controlled by a small cis-acting RE. (B) Physical monitoring of MAT a switching to MATα. The GAL::HO gene was induced for 1 h, and DNA was extracted at intervals for Southern blot analysis. The StyI restriction endonuclease cleaves in Ya but not in Yα, so that there are different restriction fragments for MAT a and MATα probed with a MAT-distal probe that also hybridizes with a common, more distal segment. In this experiment HMR was deleted (Southern blot courtesy of Neal Sugawara).
Molecular events during MAT a switching. Following HO cleavage, the ends of the DSB are resected by 5′ to 3′ exonucleases, leaving 3′ -ended ssDNA that first recruits the ssDNA binding complex, RPA, and then the Rad51 recombinase. The Rad51 nucleoprotein filament engages in a search for homologous sequences that will serve as a template for DSB repair. Rad51 promotes strand exchange and the formation of a D-loop in the Z region. PCNA-dependent DNA synthesis copies the Y sequences into the adjacent W region. Unlike normal semiconservative DNA replication where the newly synthesized strand remains base paired with its template, the new strand is displaced by 3′ to 5′ helicases, allowing it to base-pair with complementary single-stranded W sequences on the opposite side of the DSB. Removal of the nonhomologous 3 -ended segment by Rad1-Rad10 endonuclease, assisted by the mismatch repair proteins, Msh2-Msh3, allows the initiation of the DNA synthesis of the second strand. All newly synthesized DNA ends up in the recipient, leaving the donor unaltered.
Kinetics and outcomes of MAT a switching influenced by RE. Following HO cleavage, MAT a can recombine with either HMLα or with HMR α-B (BamHI). A galactose-inducible HO gene is turned on for 1 h, sufficient to cleave MAT a in nearly all cells, as shown in the Southern blots below. Cells are returned to glucose medium to prevent further HO cleavage, and the appearance of DSB repair products can be seen at 3 and 6 h. In cells with an intact RE, 90% of the switches are to MATα (i.e., using HMLα) and a few switch to MAT α-B (using HMR α-B). Fewer than 10% of the cells repair the DSB by nonhomologous end joining, restoring MAT a. When RE is deleted (reΔ), nearly all the gene conversions use HMR α-B as the donor. In the Southern blots, DNA is digested with the StyI that does not cleave in Y and with BamHI, allowing all three outcomes to be distinguished. An additional StyI fragment, distal to MAT, also hybridizes to the probe. Southern blots provided by Eric Coïc.
DNA sequence elements in the RE. RE is located in a 2.5-kb noncoding region. A minimum enhancer of about 750 bp contains five elements (A, B, C, D, and E) conserved among sensu stricto Saccharomyces spp. Regions D and E contain up to 15 repeats of the 4-bp sequence TTT(A/G). Regions A, D, and E contain binding sites for Fkh1, while the SCB sequence in element C (white box) binds Swi4 and Swi6. Region C contains an α2-Mcm1 repressor binding site that prevents other proteins from binding to RE in MATα cells. There is a second α2-Mcm1 binding site on the right side of the element. Several sterile transcripts are found in RE; their role is unknown. RE function can be obtained when the RE region is deleted and replaced by four copies of the A sequence or four or five copies of D or E.