Zhu Zhu, Chung Woo-Hyun, Shim Eun Yong, Lee Sang Eun, Ira Grzegorz
Department of Molecular & Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Cell. 2008 Sep 19;134(6):981-94. doi: 10.1016/j.cell.2008.08.037.
Formation of single-strand DNA (ssDNA) tails at a double-strand break (DSB) is a key step in homologous recombination and DNA-damage signaling. The enzyme(s) producing ssDNA at DSBs in eukaryotes remain unknown. We monitored 5'-strand resection at inducible DSB ends in yeast and identified proteins required for two stages of resection: initiation and long-range 5'-strand resection. We show that the Mre11-Rad50-Xrs2 complex (MRX) initiates 5' degradation, whereas Sgs1 and Dna2 degrade 5' strands exposing long 3' strands. Deletion of SGS1 or DNA2 reduces resection and DSB repair by single-strand annealing between distant repeats while the remaining long-range resection activity depends on the exonuclease Exo1. In exo1Deltasgs1Delta double mutants, the MRX complex together with Sae2 nuclease generate, in a stepwise manner, only few hundred nucleotides of ssDNA at the break, resulting in inefficient gene conversion and G2/M damage checkpoint arrest. These results provide important insights into the early steps of DSB repair in eukaryotes.
在双链断裂（DSB）处形成单链DNA（ssDNA）尾巴是同源重组和DNA损伤信号传导中的关键步骤。真核生物中在DSB处产生ssDNA的酶仍然未知。我们监测了酵母中可诱导DSB末端的5'链切除，并鉴定了切除两个阶段所需的蛋白质：起始和长距离5'链切除。我们表明，Mre11-Rad50-Xrs2复合物（MRX）启动5'降解，而Sgs1和Dna2降解5'链，暴露出长的3'链。删除SGS1或DNA2会减少切除以及通过远距离重复序列之间的单链退火进行的DSB修复，而剩余的长距离切除活性则取决于核酸外切酶Exo1。在exo1Δsgs1Δ双突变体中，MRX复合物与Sae2核酸酶一起逐步在断裂处仅产生几百个核苷酸的ssDNA，导致基因转换效率低下和G2/M损伤检查点停滞。这些结果为真核生物中DSB修复的早期步骤提供了重要见解。
