RAD50 is required for efficient initiation of resection and recombinational repair at random, gamma-induced double-strand break ends.
RAD50 is required for efficient initiation of resection and recombinational repair at random, gamma-induced double-strand break ends.
复制标题
Rad50是在随机的，γ诱导的双链破裂末端的有效启动和重组修复的有效启动所必需的。
DOI：
10.1371/journal.pgen.1000656
复制
发表时间：
2009-09
期刊：
影响因子：
4.5
通讯作者：
Resnick MA
中科院分区：
文献类型：
--
作者：
Westmoreland J;Ma W;Yan Y;Van Hulle K;Malkova A;Resnick MA
DNA双链断裂（DSB）末端的切除通常被认为是DSB修复途径和基因组稳定性的关键决定因素。与酶诱导的位点特异性dsb不同，人们对电离辐射等DNA损伤剂产生的随机“脏端”dsb的加工过程知之甚少。在这里，我们提出了一个新的系统来监测随机dsb修复的早期事件，基于我们的发现，在脉冲场凝胶电泳（PFGE）过程中，出芽酵母大分子末端切除产生的单链尾巴会降低迁移率。我们利用这种“PFGE-shift”来跟踪圆形染色体中单个随机DSB产生的线性分子的两端命运。在G2/M捕获的WT细胞γ辐照后10分钟内，线性化的圆形分子发生了几乎同步的pfge位移，相当于切除了几百个碱基。在辐射诱导的DSB上继续切除，因此在DSB显著修复的1小时时，每个DSB端大约有1 - 2 kb的切除。pfge的转移在WT和重组缺陷的rad52和rad51菌株中是相似的，但在exo1突变体中有所延迟。然而，在rad50和mre11零突变体中，辐射诱导的DSB末端的起始和产生在G2/M中大大减少。因此，Rad50/Mre11/Xrs2复合体负责将大多数受损末端快速加工成底物，然后进行重组修复。在非同步生长的细胞中，RAD50也有类似的需求。在rad50突变体中表现移位的少数分子中，残余切除与DSB末端的切除一致。令人惊讶的是，在辐照后1小时内，在WT和rad50中检测到双长度线性分子，但在rad52中未检测到，这可能是由于在很大程度上与切除和rad50无关的交叉。染色体DNA中的双链断裂（DSBs）是基因组变化的常见来源，可能对从酵母到人类的生物体有益或有害。虽然它们可以通过程序性细胞事件产生，但dsb通常与染色体复制缺陷有关，并且可以由各种类型的DNA损伤剂（如癌症治疗中使用的DNA损伤剂，特别是电离辐射）诱导。通过同源重组或直接连接末端，复杂的DSB识别和随后的修复系统已经发展起来。虽然对与定义的、人工产生的dsb相关的修复机制了解很多，但关于随机dsb周围事件的信息相对缺乏。使用一种新的，基于酵母的系统，适用于其他生物，我们已经解决了dsb的切除，被认为是修复的第一步。我们提供了第一个直接证据，证明细胞在γ辐射诱导的脏端具有高效的识别和起始切除系统，并且切除在很大程度上依赖于Rad50基因鉴定的Rad50/Mre11/Xrs2复合物。该系统提供了独特的机会来解决切除和修复中的其他组成部分，以及确定随机dsb和切除对其他DNA损伤剂导致的基因组不稳定性的贡献。
Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random “dirty-ended” DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE). We utilized this “PFGE-shift” to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after γ-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1–2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent. Double-strand breaks (DSBs) in chromosomal DNA are common sources of genomic change that may be beneficial or deleterious to an organism, from yeast to humans. While they can arise through programmed cellular events, DSBs are frequently associated with defective chromosomal replication, and they are induced by various types of DNA damaging agents such as those employed in cancer therapy, especially ionizing radiation. Elaborate systems have evolved for DSB recognition and subsequent repair, either by homologous recombination or by direct joining of ends. Although much is known about repair mechanisms associated with defined, artificially produced DSBs, there is a relative dearth of information about events surrounding random DSBs. Using a novel, yeast-based system that is applicable to other organisms, we have addressed resection at DSBs, considered a first step in repair. We provide the first direct evidence that cells possess a highly efficient system for recognition and initiation of resection at γ-radiation–induced dirty ends and that the resection is largely dependent on the Rad50/Mre11/Xrs2 complex, identified by the RAD50 gene. The system provides unique opportunities to address other components in resection and repair as well as to identify the contribution of random DSBs and resection to genome instability resulting from other DNA damaging agents.
登录
查看更多内容
影响因子：
11.4
作者：
Aboussekhra, A;Vialard, JE;Lowndes, NF
通讯作者：
Lowndes, NF
影响因子：
16
作者：
Lengsfeld, Bettina M.;Rattray, Alison J.;Paull, Tanya T.
通讯作者：
Paull, Tanya T.
映射减数分裂的单链DNA揭示了酿酒酵母中DNA双链断裂的新景观。
影响因子：
9.8
作者：
Buhler, Cyril;Borde, Valerie;Lichten, Michael
通讯作者：
Lichten, Michael
影响因子：
16
作者：
Lao, Jessica P.;Oh, Steve D.;Hunter, Neil
通讯作者：
Hunter, Neil
影响因子：
3.9
作者：
BEAM, CA;MORTIMER, RK;TOBIAS, CA
通讯作者：
TOBIAS, CA