SIRF: Quantitative in situ analysis of protein interactions at DNA replication forks.
SIRF: Quantitative in situ analysis of protein interactions at DNA replication forks.
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DOI：
10.1083/jcb.201709121
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发表时间：
2018-04-02
期刊：
影响因子：
--
通讯作者：
Schlacher K
中科院分区：
文献类型：
--
作者：
Roy S;Luzwick JW;Schlacher K
Roy等人。描述一种新的方法来测量活跃和停滞的DNA复制叉处的直接蛋白质关联，称为DNA复制叉处的蛋白质相互作用的原位分析。EDU-CHASE、点击-化学和聚乳酸-复合体系具有定量、灵敏、有效、单胞分辨的特点，适用于伴随的多参数分析。DNA复制反应是多种细胞过程的核心，包括发育、癌症病因、药物治疗和耐药性。存在许多蛋白质和途径来确保DNA复制的保真度和对停滞或损坏的复制叉子的保护。一直以来，参与DNA复制的蛋白质突变与各种疾病有关，包括胚胎发育和免疫缺陷、加速衰老、炎症增加、血液疾病和癌症。因此，对活跃和停滞的复制叉处的蛋白质相互作用进行有效的定量分析的工具是先进和准确的生物学理解的关键。在这里，我们描述了一个灵敏的单细胞水平的测试系统，用于定量分析蛋白质与新生DNA的相互作用。具体地说，我们使用邻近连接与5‘-乙烯-2’-脱氧尿苷点击化学相结合的方法，实现了对DNA复制叉处蛋白质相互作用的稳健的原位分析，适合于异质细胞群体的多参数分析。我们提供灵敏度、准确性、接近性和量化方面的验证数据。利用SIRF，我们对53BP1在瞬时停滞的复制叉处对途径选择的调控获得了新的见解。
Roy et al. describe a novel assay to measure direct protein associations at active and stalled DNA replication forks, called in situ analysis of protein interactions at DNA replication forks. The EdU-chase, click-chemistry, and PLA-composite system is quantitative, sensitive, and effective, with single-cell resolution suitable for concomitant multiparameter analysis. DNA replication reactions are central to diverse cellular processes including development, cancer etiology, drug treatment, and resistance. Many proteins and pathways exist to ensure DNA replication fidelity and protection of stalled or damaged replication forks. Consistently, mutations in proteins involved in DNA replication are implicated in diverse diseases that include defects during embryonic development and immunity, accelerated aging, increased inflammation, blood disease, and cancer. Thus, tools for efficient quantitative analysis of protein interactions at active and stalled replication forks are key for advanced and accurate biological understanding. Here we describe a sensitive single-cell–level assay system for the quantitative analysis of protein interactions with nascent DNA. Specifically, we achieve robust in situ analysis of protein interactions at DNA replication forks (SIRF) using proximity ligation coupled with 5′-ethylene-2′-deoxyuridine click chemistry suitable for multiparameter analysis in heterogeneous cell populations. We provide validation data for sensitivity, accuracy, proximity, and quantitation. Using SIRF, we obtained new insight on the regulation of pathway choice by 53BP1 at transiently stalled replication forks.
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影响因子：
64.8
作者：
Ray Chaudhuri A;Callen E;Ding X;Gogola E;Duarte AA;Lee JE;Wong N;Lafarga V;Calvo JA;Panzarino NJ;John S;Day A;Crespo AV;Shen B;Starnes LM;de Ruiter JR;Daniel JA;Konstantinopoulos PA;Cortez D;Cantor SB;Fernandez-Capetillo O;Ge K;Jonkers J;Rottenberg S;Sharan SK;Nussenzweig A
通讯作者：
Nussenzweig A
影响因子：
16.8
作者：
Davies, Sally L.;North, Phillip S.;Hickson, Ian D.
通讯作者：
Hickson, Ian D.
影响因子：
56.9
作者：
Michalet, X;Ekong, R;Bensimon, A
通讯作者：
Bensimon, A
DOI：
10.1038/nrm2851
发表时间：
2010-03
期刊：
Nature reviews. Molecular cell biology
影响因子：
--
作者：
通讯作者：
--
DOI：
10.1083/jcb.201011083
发表时间：
2011-04-04
期刊：
The Journal of cell biology
影响因子：
--
作者：
Harrigan JA;Belotserkovskaya R;Coates J;Dimitrova DS;Polo SE;Bradshaw CR;Fraser P;Jackson SP
通讯作者：
Jackson SP