Regulation Of Non-homologous End Joining Research Articles

NEJ1 controls non-homologous end joining in Saccharomyces cerevisiae.

Broken DNA ends are rejoined by non-homologous end-joining (NHEJ) pathways requiring the Ku proteins (Ku70, Ku80), DNA ligase IV and its associated protein Lif1/Xrcc4 (ref. 1). In mammalian meiotic cells, Ku protein levels are much lower than in somatic cells, apparently reducing the capacity of meiotic cells to carry out NHEJ and thereby promoting homologous recombination. In Saccharomyces cerevisiae, NHEJ is also downregulated in meiosis-competent MATa/MAT alpha diploid cells in comparison with diploids or haploids expressing only MATa or MAT alpha. Diploids expressing both MATa and MAT alpha show enhanced mitotic homologous recombination. Here we report that mating-type-dependent regulation of NHEJ in budding yeast is caused in part by transcriptional repression of both LIF1 and the gene NEJ1 (YLR265C)--identified from microarray screening of messenger RNAs. Deleting NEJ1 reduces NHEJ 100-fold in MATa or MAT alpha haploids. Constitutive expression of NEJ1, but not expression of LIF1, restores NHEJ in MATa/MAT alpha cells. Nej1 regulates the subcellular distribution of Lif1. A green fluorescent protein (GFP)-Lif1 fusion protein accumulates in the nucleus in cells expressing NEJ1 but is largely cytoplasmic when NEJ1 is repressed.

NHEJ regulation by mating type is exercised through a novel protein, Lif2p, essential to the ligase IV pathway.

In the yeast Saccharomyces cerevisiae, DNA double strand break (DSB) repair by nonhomologous end-joining (NHEJ) requires the DNA end-binding heterodimer Yku70p-Yku80p and the ligase Dnl4p associated with its cofactor Lif1p. NHEJ efficiency is down-regulated in MATa/MATalpha cells relative to MATa or MATalpha cells, but the mechanism of this mating type regulation is unknown. Here we report the identification of Lif2p, a S. cerevisiae protein that interacts with Lif1p in a two-hybrid system. Disruption of LIF2 abolishes the capacity of cells to repair DSBs by end-joining to the same extent than lif1 and dnl4 mutants. In MATa/MATalpha cells, Lif2p steady-state level is strongly repressed when other factors involved in NHEJ are unaffected. Increasing the dosage of the Lif2p protein can suppress the NHEJ defect in a/alpha cells. Together, these results indicate that NHEJ regulation by mating type is achieved, at least in part, by a regulation of Lif2p activity.

Nej1p, a cell type-specific regulator of nonhomologous end joining in yeast

Mutant yeast strains lacking the silencing proteins Sir2p, Sir3p, or Sir4p have a defect in a DNA double-strand break (DSB) repair pathway, called nonhomologous end joining (NHEJ) [1, 2]. Mutations in sir genes also lead to the simultaneous expression of a and α mating type information, thus generating a nonmating haploid cell type [3] with many properties shared with a/α diploids. We addressed whether cell type or Sir proteins per se regulate NHEJ by investigating the role of a novel haploid-specific gene in NHEJ. This gene, NEJ1, was required for efficient NHEJ, and transcription of NEJ1 was completely repressed in a/α diploid [4] and sir haploid strains. The NEJ1 promoter contained a consensus binding site for the a1/α2 repressor, explaining the cell type-specific expression. Expression of Nej1p from a constitutive promoter in a/α diploid and sir mutant strains completely rescued the defect in NHEJ, thus showing that Sir proteins per se were dispensable for NHEJ. Nej1p and Lif1P[5], the yeast XRCC4 homolog, interacted in two independent assays, and Nej1p localized to the nucleus, suggesting that Nej1p may have a direct role in NHEJ.