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Non-homologous end-joining (NHEJ) is required in mammals both for V (D)J recombination2 and for repairing double-stranded DNA breaks. NHEJ also occurs in yeast3,4, and it has been reported that Sir proteins are required for this process5,6. 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/α-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 double-stranded break in vector sequences lacking homology to the yeast genome were transformed into yeast. The frequency of transformants was used as a measure of NHEJ5,6. Results obtained from SIR+ and sir− strains were consistent with previous findings5,6. NHEJ in sir− strains was 20-fold less efficient than in wild-type strains (Table 1). However, assays performed in SIR+ and sir− strains in which all mating-type genes had been inactivated by a promoter deletion (hml aΔp mataΔp hmraΔp, abbreviated here as a−a−a−) revealed that the absence of mating-type heterozygosity suppressed the defect in NHEJ exhibited by the sir− strains (Table 1).

Table 1 Efficiency of non-homologous end-joining in haploid and diploid strains
Full size table

We performed plasmid-rejoining assays on two SIR+diploid strains, an a/α diploid and a non-a/α diploid ( mataδp/MATα, in which only α information is expressed). The non-a/α diploid strain accomplished NHEJ tenfold more efficiently than the a/α 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/α cells indicates that a gene required for NHEJ was regulated by the a1/α2 repressor. RNA blot analysis of HDF1, HDF2, DNL4, XRS2 and MRE11 , the leading candidate genes7,8,9,10 in wild-type, sir3, a−a−a−and a− a−a− 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/α2 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/α 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 double-stranded break, NHEJ could lead to exchange-type aberrations11, indicating that homology-driven repair should be the preferred pathway. But haploid cells in the G1 phase of the cell cycle lack homologues and so rely on NHEJ. With NHEJ under the control of the a1/α2 repressor, a yeast cell could adapt the repair process, using the NHEJ pathway primarily when homology-driven repair is not possible. This would require an a1/α2-repressed gene that is important for NHEJ. Alternatively, a1/α2 could inhibit NHEJ indirectly by upregulating the RAD52 homologous repair pathway to outcompete the NHEJ pathway for the repair of double-stranded breaks.