Visualization of human Bloom's syndrome helicase molecules bound to homologous recombination intermediates
Visualization of human Bloom's syndrome helicase molecules bound to homologous recombination intermediates
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DOI：
10.1096/fj.13-234088
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发表时间：
2013-12-01
期刊：
影响因子：
4.8
通讯作者：
Kovacs, Mihaly
中科院分区：
文献类型：
--
作者：
Gyimesi, Mate;Pires, Ricardo H.;Kovacs, Mihaly
同源重组（HR）是双链DNA断裂（DSB）修复的关键过程，可引发癌症或细胞死亡。人类Bloom综合征RecQ家族DNA解旋酶（BLM）具有促进DSB修复和避免不合理HR的复杂活性，其寡聚体组装状态一直是其活性中一个尚未解决的关键问题。在这项研究中，我们评估了BLM的结构和低聚状态，在关键HR-中间DNA结构的存在和不存在下，通过使用单分子可视化（电子显微镜和原子力显微镜单粒子分析）和溶液生物物理（动态光散射，动力学和平衡结合）技术。除了全长BLM，我们使用了以前的特点截短的构建体（BLM 642 -1290）作为单体控制。相反，以前的模型提出了一个成环的低聚物，我们发现大多数的BLM分子是单体在所有检查的条件。然而，BLM表现出倾向于形成二聚体时，绑定到分支HR中间体。我们的研究结果表明，人力资源活动需要单链DNA易位进行单体BLM，而复杂的DNA结构遇到和溶解的BLM在后期阶段的人力资源诱导部分寡聚化的解旋酶。
Homologous recombination (HR) is a key process in the repair of double-stranded DNA breaks (DSBs) that can initiate cancer or cell death. Human Bloom's syndrome RecQ-family DNA helicase (BLM) exerts complex activities to promote DSB repair while avoiding illegitimate HR. The oligomeric assembly state of BLM has been a key unresolved aspect of its activities. In this study we assessed the structure and oligomeric state of BLM, in the absence and presence of key HR-intermediate DNA structures, by using single-molecule visualization (electron microscopic and atomic force microscopic single-particle analysis) and solution biophysical (dynamic light scattering, kinetic and equilibrium binding) techniques. Besides full-length BLM, we used a previously characterized truncated construct (BLM642-1290) as a monomeric control. Contrary to previous models proposing a ring-forming oligomer, we found the majority of BLM molecules to be monomeric in all examined conditions. However, BLM showed a tendency to form dimers when bound to branched HR intermediates. Our results suggest that HR activities requiring single-stranded DNA translocation are performed by monomeric BLM, while complex DNA structures encountered and dissolved by BLM in later stages of HR induce partial oligomerization of the helicase.