Abstract
During Saccharomyces cerevisiae mating-type switching, an HO endonuclease-induced double-strand break (DSB) at MAT is repaired by recombining with one of two donors, HMLα or HMR a, located at opposite ends of chromosome III. MAT a cells preferentially recombine with HMLα; this decision depends on the Recombination Enhancer (RE), located about 17 kb to the right of HML. In MATα cells, HML is rarely used and RE is bound by the MATα2-Mcm1 corepressor, which prevents the binding of other proteins to RE. In contrast, in MAT a cells, RE is bound by multiple copies of Fkh1 and a single copy of Swi4/Swi6. We report here that, when RE is replaced with four LexA operators in MAT a cells, 95% of cells use HMR for repair, but expression of a LexA-Fkh1 fusion protein strongly increases HML usage. A LexA-Fkh1 truncation, containing only Fkh1's phosphothreonine-binding FHA domain, restores HML usage to 90%. A LexA-FHA-R80A mutant lacking phosphothreonine binding fails to increase HML usage. The LexA-FHA fusion protein associates with chromatin in a 10-kb interval surrounding the HO cleavage site at MAT, but only after DSB induction. This association occurs even in a donorless strain lacking HML. We propose that the FHA domain of Fkh1 regulates donor preference by physically interacting with phosphorylated threonine residues created on proteins bound near the DSB, thus positioning HML close to the DSB at MAT. Donor preference is independent of Mec1/ATR and Tel1/ATM checkpoint protein kinases but partially depends on casein kinase II. RE stimulates the strand invasion step of interchromosomal recombination even for non-MAT sequences. We also find that when RE binds to the region near the DSB at MAT a then Mec1 and Tel1 checkpoint kinases are not only able to phosphorylate histone H2A (γ-H2AX) around the DSB but can also promote γ-H2AX spreading around the RE region.
Highlights
Saccharomyces mating-type switching occurs through a DSBinitiated intrachromosomal gene conversion event at MAT, using one of two donors on chromosome III, HML and HMR (Figure 1A) [1,2,3]
Donor preference is governed by the Recombination Enhancer (RE), located about 17 kb from HML
Donor preference depends on the phosphothreonine-binding FHA domain of Fkh1
Summary
Saccharomyces mating-type switching occurs through a DSBinitiated intrachromosomal gene conversion event at MAT, using one of two donors on chromosome III, HML and HMR (Figure 1A) [1,2,3]. Switching is initiated by expression of the site-specific HO endonuclease that cleaves only one site in the yeast genome, MATa or MATa. The unexpressed mating-type genes in HMLa and HMRa contain HO cleavage sites, but they are not cut because these regions are heterochromatic [4,5,6]. Either HMLa or HMRa can be used to repair a DSB at MAT, there is a strong mating type-dependent preference for the choice of the two donors. Donor preference is not altered if the mating-type genes encoded in the Y region are changed, e.g. if HMR carries Ya instead of Ya or if HML is replaced with HMR [7,8]
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