Yeast cell-type regulation of DNA repair

Abstract

cryptic a and Ȋ genes resident at the HML and HMR loci. As a result, sir mutant strains have the properties of a/Ȋ diploids.Non-homologous end-joining (NHEJ) is required in mammals both for V(D)J recombination 2 and for repairing doublestranded DNA breaks. NHEJ also occurs in yeast 3,4 , and it has been reported that Sir proteins are required for this process 5,6 . This observation was interpreted to mean that Sir proteins are involved directly in NHEJ, perhaps by forming a heterochromatin-like structure at double-stranded breaks. But we have found evidence for an alternative interpretation: that the a/Ȋ-state regulates NHEJ and that sir mutations affect NHEJ indirectly.To distinguish between these two possibilities, we performed plasmid-rejoining assays. Plasmids that were linearized by restriction enzymes and contained a doublestranded break in vector sequences lacking homology to the yeast genome were transformed into yeast. The frequency of transformants was used as a measure of NHEJ 5,6 . Results obtained from SIR + and sir ǁ strains were consistent with previous findings 5,6 . NHEJ in sir ǁ strains was 20-fold less efficient than in wild-type strains (Table 1). However, assays performed in SIR + and sir ǁ strains in which all mating-type genes had been inactivated by a promoter deletion (hmla∆p mata∆p hmra∆p, abbreviated here as a ǁ a ǁ a ǁ ) revealed that the absence of mating-type heterozygosity suppressed the defect in NHEJ exhibited by the sir ǁ strains (Table 1).We performed plasmid-rejoining assays on two SIR + diploid strains, an a/Ȋ diploid and a non-a/Ȋ diploid (mata∆p/MATȊ, in which only Ȋ information is expressed). The non-a/Ȋ diploid strain accomplished NHEJ tenfold more efficiently than the a/Ȋ diploid (Table 1). NHEJ was therefore controlled by mating-type heterozygosity, and no cell-type-independent effect of sir mutations was detected.The defect in NHEJ found in a/Ȋ cells indicates that a gene required for NHEJ was regulated by the a1/Ȋ2 repressor. RNA blot analysis of HDF1, HDF2, DNL4, XRS2 and MRE11, the leading candidate genes 7-10 in wild-type, sir3, a ǁ a ǁ a ǁ and a ǁ a ǁ a ǁ sir3 strains, revealed that all five genes were comparably expressed in SIR3 and sir3 strains (data not shown). These genes are therefore not relevant targets for the a1/Ȋ2 repression of NHEJ.Our results provide evidence against a direct role for heterochromatin formation in NHEJ, indicating instead that the efficiency of NHEJ is controlled by cell type. But our data do not exclude the possibility that different strains might yield different results: indeed, the W303 strain we used contains a mild rad5 mutation. However, the a/Ȋ regulation of NHEJ found here can explain problems associated with DNA repair in yeast. Diploid cells that suffer a double-stranded break have a homologous partner that can perform a homologydriven recombinational repair process. In cells that have more than one doublestranded break, NHEJ could lead to exchange-type aberrations 11 , indicating that homology-driven repair should be the pre...

Non-homologous end-joining factors ofSaccharomyces cerevisiae

“…In such cells, homology-driven repair is impossible and therefore NHEJ may be required. This is consistent with the findings that showed that NHEJ efficiency is increased in stationary [152] and haploid cells [153] (see sections below) compared to exponentially and diploid cells, respectively. Notably, the lig4 or lif1 mutant is not UV sensitive [40][41][42]44].…”

Bypassing the Requirement for an Essential MYST Acetyltransferase

“…The assay consists of transforming a linearized plasmid and counting the transformants that repaired the linear plasmid to convert it into a circular and thereby stably maintained plasmid (Åström et al 1999;Lee et al 1999). We found that esa1-414 and esa1Δ sds3Δ strains had increased NHEJ repair efficiency compared to wild type, one possible reason for their DNA damage sensitivity phenotypes ( Figure 5B).…”

“…Assays with pRS316 were performed with established methods (Åström et al 1999;Lee et al 1999). Four independent experiments were performed, and P-values were calculated using the Student's t-test.…”

“…NHEJ is error-prone as it predominantly ligates broken DNA ends (Betermier et al 2014), whereas the HR pathway is more accurate as it uses an intact copy of the affected region as a template for repair. The a1-a2 repressor expressed in diploid and SIR2 null strains represses specific genes involved in the NHEJ pathway (Åström et al 1999;Valencia et al 2001). This is believed to increase HR efficiency, as diploid cells contain a second copy of each chromosome that can be used as a template for repair (Haber 2012).…”

Restricting the ligation step of non-homologous end-joining

“…A first indication that NHEJ is under active control in S. cerevisiae appeared in the late 1990s when it was demonstrated that this process is down-regulated by Mata1-Matα2, resulting in less efficient NHEJ in diploid cells compared to haploid cells [7,8]. Subsequently, the responsible target protein, Nej1, was identified [9][10][11][12] and shown to be encoded by a haploidspecific gene, expression of which is virtually undetectable in the presence of Mata1-Matα2 because of a consensus-binding site for this repressor in the NEJ1 promoter [9,10].…”