Donor Preference Associated with MAT Switching

There is one other remarkable aspect of MAT switching: the mating-type regulated choice of HML or HMR as a donor in repairing the HO-induced DSB. A MATa haploid recombines with HMLa about 90% of the time, whereas a


MA 7a """ HO cleavage

5' to 3' resection

Strand invasion (RPA, RadSl)

Primer extension (PCNA and I'olS and e)

3' nonhomoiogv removal (Radl-RadlO; \Isli2-Msli3)

Strand displacement Second strand synthesis


Fig. 2. Mechanism of MATa switching. Following the creation of an HO-induced DSB, the 5' ends are resected to create 3'-ended single-stranded tails that are bound first by the single-stranded binding protein complex RPA and then by the Rad51 recombinase that promotes strand invasion of the Z region of the HMLa donor. The 3' end of the invading DNA is used as a primer to initiate new DNA synthesis. The newly synthesized strand is apparently displaced and pairs with resected DNA on the other side of the DSB. MAT switching is completed, almost always without an accompanying crossover, by copying the second strand

MATa haploid selectively chooses HMRa. This ensures that, most of the time, the process of switching will produce an equal number of juxtaposed cells of opposite mating type after two cell divisions. "Donor preference" is not determined by the differences between the silencer regions surrounding HML and HMR, nor by the Ya or Ya sequences themselves; in a strain in which HMLa is deleted and replaced by a cloned HMRa locus, MATa cells continue to select the left-arm donor (Weiler and Broach 1992). Control of donor preference lies with the recombination enhancer (RE), a small, cis-acting sequence located 17 kb away from HML (Wu and Haber 1996; Wu et al. 1998). In MATa cells, RE is active and facilitates the use of HML over HMR. In MATa cells, RE is repressed, and the left-arm donor becomes in some way inaccessible; consequently, the right-arm donor is used preferentially. RE acts over


MA 7a """ HO cleavage the entire 115 kb of the left arm of chromosome III (Wu and Haber 1996; Sun et al. 2002). A donor placed anywhere along this arm can be used selectively in MATa cells. When RE is deleted, the left-arm donor becomes inaccessible in MATa cells. Most of the activity of RE is contained in a segment of about 750 bp. Further analysis, including comparisons between the REs of three Sac-charomyces species (Wu et al. 1998) and the creation of synthetic RE sequences from multimers of conserved subregions (Wu and Haber 1996; Sun et al. 2002), showed that RE consists of several highly conserved domains, including two regions with ten or more iterations of TTT(A/G) and a highly conserved MAT "Greek alpha" 2-Mcml "operator region" which shares strong sequence identity with sequences that control expression of "a-specific genes".

Repression of RE in MATa cells occurs by binding of the a2-Mcml repressor that also turns off a-specific genes to the operator site and leads to the establishment of highly positioned nucleosomes across the 2.5-kb region containing RE (there are no open reading frames in this region; there is no change in the chromatin structure of any of the genes along the chromosome arm nor changes in the silencing of HML). In MATa, the positioned nucleosomes are absent and several DNasel-hypersensitive sites indicative of protein binding are found (Wu et al. 1998). Activation of RE depends on Mcml (which is also an activator of expression of a-specific genes). Mcml binding facilitates the binding of the Fkhl transcription regulator (Sun et al. 2002). The complex RE sequences can be replaced by as few as four 22-bp copies of one of the Fkhl-binding sites located in several conserved subregions of the RE. Small effects on MATa's use of HML have been seen when the cohesin assembly protein Chll is deleted (Weiler et al. 1995).

How RE acts remains unknown, but experiments recruiting the LacI-GFP fusion protein to LacO arrays inserted near HML, MAT or HMR have provided some clues. Live-cell imaging of GFP-tagged chromosomes suggests that the left arm of chromosome III is more confined in MATa (or RE-deleted) cells than in MATa (Bressan et al. 2004). The molecular basis of this tethering remains under investigation.

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