Lewis L K, Westmoreland J W, Resnick M A
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Genetics. 1999 Aug;152(4):1513-29. doi: 10.1093/genetics/152.4.1513.
Repair of double-strand breaks (DSBs) in chromosomal DNA by nonhomologous end-joining (NHEJ) is not well characterized in the yeast Saccharomyces cerevisiae. Here we demonstrate that several genes associated with NHEJ perform essential functions in the repair of endonuclease-induced DSBs in vivo. Galactose-induced expression of EcoRI endonuclease in rad50, mre11, or xrs2 mutants, which are deficient in plasmid DSB end-joining and some forms of recombination, resulted in G2 arrest and rapid cell killing. Endonuclease synthesis also produced moderate cell killing in sir4 strains. In contrast, EcoRI caused prolonged cell-cycle arrest of recombination-defective rad51, rad52, rad54, rad55, and rad57 mutants, but cells remained viable. Cell-cycle progression was inhibited in excision repair-defective rad1 mutants, but not in rad2 cells, indicating a role for Rad1 processing of the DSB ends. Phenotypic responses of additional mutants, including exo1, srs2, rad5, and rdh54 strains, suggest roles in recombinational repair, but not in NHEJ. Interestingly, the rapid cell killing in haploid rad50 and mre11 strains was largely eliminated in diploids, suggesting that the cohesive-ended DSBs could be efficiently repaired by homologous recombination throughout the cell cycle in the diploid mutants. These results demonstrate essential but separable roles for NHEJ pathway genes in the repair of chromosomal DSBs that are structurally similar to those occurring during cellular development.
在酿酒酵母中，非同源末端连接（NHEJ）对染色体DNA双链断裂（DSB）的修复作用尚未得到充分研究。在此，我们证明了几个与NHEJ相关的基因在体内对核酸内切酶诱导的DSB修复中发挥着重要作用。在缺乏质粒DSB末端连接和某些形式重组的rad50、mre11或xrs2突变体中，半乳糖诱导EcoRI核酸内切酶表达会导致G2期停滞和细胞快速死亡。核酸内切酶的合成在sir4菌株中也会导致适度的细胞死亡。相比之下，EcoRI会使重组缺陷型的rad51、rad52、rad54、rad55和rad57突变体的细胞周期长时间停滞，但细胞仍能存活。切除修复缺陷型的rad1突变体细胞周期进程受到抑制，但rad2细胞不受影响，这表明Rad1在DSB末端加工中发挥作用。包括exo1、srs2、rad5和rdh54菌株在内的其他突变体的表型反应表明它们在重组修复中发挥作用，但在NHEJ中不起作用。有趣的是，单倍体rad50和mre11菌株中的快速细胞死亡在二倍体中基本消除，这表明在二倍体突变体的整个细胞周期中，粘性末端的DSB可以通过同源重组有效修复。这些结果证明了NHEJ途径基因在修复与细胞发育过程中发生的结构相似的染色体DSB中起着重要但可分离的作用。
