Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination.
Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination.
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DOI：
10.1073/pnas.2021963118
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发表时间：
2021-03-16
影响因子：
11.1
通讯作者：
Rothenberg E
中科院分区：
文献类型：
--
作者：
Whelan DR;Rothenberg E
DNA切除是DNA双链断裂（DSB）同源重组修复途径中的初始决定性步骤。然而，在这一过程中的关键蛋白质的个别作用和串扰仍不清楚。为了解决这个复杂的过程的时空动力学，我们应用单分子定位显微镜沿着与强大的分析方法。我们的数据揭示了MRE 11，BRCA 1和CtIP作为多聚体和单独的动态作用和相互作用; 53 BP 1的招募和空间排斥; BLM解旋酶与EXO 1和DNA 2核酸酶的作用;以及MRE 11外切酶/内切酶抑制的抑制机制。总之，我们的研究结果描述了DNA DSB修复的重要方面，并强调了簇状损伤修复和散发性内源性断裂之间的关键差异。同源重组（HR）是DNA双链断裂（DSB）修复的主要途径。驱动HR过程的初始步骤是在DSB处切除DNA，在此期间，大量核酸酶、介质和信号蛋白以仍然难以捉摸的方式在损伤灶处积累。使用单分子定位超分辨率（SR）成像分析，我们专门可视化的时空行为的关键介质和核酸酶蛋白，因为他们切除DNA在单端双链断裂（seDSB）形成在折叠复制叉。通过表征这些协会，我们揭示了在体内动力学的切除复合物参与生成的长单链DNA（ssDNA）的悬垂之前同源搜索。我们发现，53 BP 1，一种已知拮抗HR的蛋白质，在早期切除期间被招募到seDSB病灶，但在空间上与修复活动分离。同时，CtBP相互作用蛋白（CtIP）和MRN（MRE 11-RAD 51-NBS 1）与seDSB结合，相互作用并与BRCA 1相互作用。HR核酸酶EXO 1和DNA 2也被募集并彼此共定位，并与修复解旋酶布卢姆综合征蛋白（BLM）共定位，表明多个同时切除事件。复制蛋白A（RPA）积累和ssDNA生成的定量显示，在断裂诱导后2至4小时完成切除。然而，BRCA 1和BLM都持续到HR后期，表明在同源性搜索和修复解析中具有潜在作用。此外，我们表明，BRCA 1的初始招聘和Ku的去除在很大程度上是独立的MRE 11核酸外切酶活性，但依赖于MRE 11核酸内切酶活性。综合起来，我们的观察提供了HR修复期间切除的详细描述。
DNA resection is an initial, decisive step in the homologous recombination repair pathway of DNA double-strand breaks (DSBs). However, the individual roles and cross-talk of key proteins in this process remain unclear. To resolve the spatiotemporal dynamics of this intricate process, we applied multicolor single-molecule localization microscopy along with robust analytical approaches. Our data reveal the dynamic actions and interactions of MRE11, BRCA1, and CtIP both as a multimer and individually; the recruitment and spatial exclusion of 53BP1; the role of BLM helicase alongside EXO1 and DNA2 nucleases; and the inhibitory mechanisms of MRE11 exo-/endonuclease inhibition. Together, our findings describe important aspects of DNA DSB repair and highlight key differences between repair of clustered damage and sporadic endogenous breaks. Homologous recombination (HR) is a major pathway for repair of DNA double-strand breaks (DSBs). The initial step that drives the HR process is resection of DNA at the DSB, during which a multitude of nucleases, mediators, and signaling proteins accumulates at the damage foci in a manner that remains elusive. Using single-molecule localization super-resolution (SR) imaging assays, we specifically visualize the spatiotemporal behavior of key mediator and nuclease proteins as they resect DNA at single-ended double-strand breaks (seDSBs) formed at collapsed replication forks. By characterizing these associations, we reveal the in vivo dynamics of resection complexes involved in generating the long single-stranded DNA (ssDNA) overhang prior to homology search. We show that 53BP1, a protein known to antagonize HR, is recruited to seDSB foci during early resection but is spatially separated from repair activities. Contemporaneously, CtBP-interacting protein (CtIP) and MRN (MRE11-RAD51-NBS1) associate with seDSBs, interacting with each other and BRCA1. The HR nucleases EXO1 and DNA2 are also recruited and colocalize with each other and with the repair helicase Bloom syndrome protein (BLM), demonstrating multiple simultaneous resection events. Quantification of replication protein A (RPA) accumulation and ssDNA generation shows that resection is completed 2 to 4 h after break induction. However, both BRCA1 and BLM persist later into HR, demonstrating potential roles in homology search and repair resolution. Furthermore, we show that initial recruitment of BRCA1 and removal of Ku are largely independent of MRE11 exonuclease activity but dependent on MRE11 endonuclease activity. Combined, our observations provide a detailed description of resection during HR repair.
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影响因子：
19
作者：
Ceccaldi R;Rondinelli B;D'Andrea AD
通讯作者：
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作者：
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通讯作者：
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通讯作者：
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