Regulation of Budding Yeast Mating-Type Switching Donor Preference by the FHA Domain of Fkh1

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.

Chromosome position determines the success of double-strand break repair

“…The assay was modified from the PCR-based donor preference assay described previously (25). In brief, cells were grown in YP-Lactate medium to log phase in the way described in the viability assay above.…”

“…6B). The use of a poor donor can also be improved by placing an ∼800-bp segment containing the cis-acting recombination enhancer (RE) sequence (24,25) ∼5 kb from the donor (Fig. 6C).…”

Mating-Type Genes andMATSwitching inSaccharomyces cerevisiae

“…The RE contains no open reading frame, but indeed there are two "sterile" transcripts of the RE region that are transcribed in MATa, but not MATa . Despite the increasing evidence of the role of noncoding RNAs in regulating chromatin structure in organisms that also have RNAi silencing, it is unlikely that the sequence of the RNA transcript is important for RE activity, as truncations of RE that remove all of this transcribed region have full activity Wu et al 1998;Li et al 2012). Indeed, as we will see below, the entire RE can be replaced by completely foreign, LexA-binding domains and RE activity can be mimicked by the binding of a LexA-Fkh1 protein.…”

A Life Investigating Pathways That Repair Broken Chromosomes

“…RE contains multiple binding sites for the Fkh1 transcription factor, but it is only the phosphothreonine binding domain of Fkh1 that is required. When RE is replaced by four LexA binding sites, a LexA-Fkh1 fusion protein mimics the normal RE function and a LexA-FHA Fkh1 construct has full activity (76,133). ChIP experiments show that RE binds close to DSB ends and GFP-FHA Fkh1 fusion protein localizes to multiple HO-induced DSBs (C.S.…”