Lewis L Kevin, Karthikeyan G, Westmoreland James W, Resnick Michael A
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Genetics. 2002 Jan;160(1):49-62. doi: 10.1093/genetics/160.1.49.
Rad50, Mre11, and Xrs2 form a nuclease complex that functions in both nonhomologous end-joining (NHEJ) and recombinational repair of DNA double-strand breaks (DSBs). A search for highly expressed cDNAs that suppress the DNA repair deficiency of rad50 mutants yielded multiple isolates of two genes: EXO1 and TLC1. Overexpression of EXO1 or TLC1 increased the resistance of rad50, mre11, and xrs2 mutants to ionizing radiation and MMS, but did not increase resistance in strains defective in recombination (rad51, rad52, rad54, rad59) or NHEJ only (yku70, sir4). Increased Exo1 or TLC1 RNA did not alter checkpoint responses or restore NHEJ proficiency, but DNA repair defects of yku70 and rad27 (fen) mutants were differentially suppressed by the two genes. Overexpression of Exo1, but not mutant proteins containing substitutions in the conserved nuclease domain, increased recombination and suppressed HO and EcoRI endonuclease-induced killing of rad50 strains. exo1 rad50 mutants lacking both nuclease activities exhibited a high proportion of enlarged, G2-arrested cells and displayed a synergistic decrease in DSB-induced plasmid:chromosome recombination. These results support a model in which the nuclease activity of the Rad50/Mre11/Xrs2 complex is required for recombinational repair, but not NHEJ. We suggest that the 5'-3' exo activity of Exo1 is able to substitute for Rad50/Mre11/Xrs2 in rescission of specific classes of DSB end structures. Gene-specific suppression by TLC1, which encodes the RNA subunit of the yeast telomerase complex, demonstrates that components of telomerase can also impact on DSB repair pathways.
Rad50、Mre11和Xrs2形成一种核酸酶复合物，该复合物在DNA双链断裂（DSB）的非同源末端连接（NHEJ）和重组修复中均发挥作用。对可抑制rad50突变体DNA修复缺陷的高表达cDNA进行筛选，得到了两个基因的多个分离株：EXO1和TLC1。EXO1或TLC1的过表达增加了rad50、mre11和xrs2突变体对电离辐射和甲磺酸甲酯（MMS）的抗性，但未增加重组缺陷菌株（rad51、rad52、rad54、rad59）或仅NHEJ缺陷菌株（yku70、sir4）的抗性。Exo1或TLC1 RNA的增加并未改变检查点反应或恢复NHEJ能力，但这两个基因对yku70和rad27（fen）突变体的DNA修复缺陷有不同程度的抑制作用。Exo1的过表达而非在保守核酸酶结构域中含有替代突变的蛋白质，增加了重组并抑制了HO和EcoRI内切核酸酶诱导的rad50菌株的杀伤作用。缺乏两种核酸酶活性的exo1 rad50突变体表现出高比例的增大、G2期阻滞细胞，并且在DSB诱导的质粒：染色体重组中呈现协同下降。这些结果支持了一种模型，即Rad50/Mre11/Xrs2复合物的核酸酶活性是重组修复所必需的，但不是NHEJ所必需的。我们认为，Exo1的5'-3'外切核酸酶活性能够在特定类型的DSB末端结构的切除中替代Rad50/Mre11/Xrs2。由编码酵母端粒酶复合物RNA亚基的TLC1进行的基因特异性抑制表明，端粒酶的组分也可影响DSB修复途径。
