Roy Sunetra, Schlacher Katharina
Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Bio Protoc. 2019 Sep 20;9(18):e3377. doi: 10.21769/BioProtoc.3377.
The duplication of DNA is a fundamental process that is required for the transfer of the genetic information from parent to daughter cells. Aberrant DNA replication processes are associated with diverse disease phenotypes, including developmental defects, ageing disorders, blood disorders such as Fanconi Anemia, increased inflammation and cancer. Therefore, the development of tools to study proteins associated with error-free DNA replication processes is of paramount importance. So far, methods to study proteins associated with nascent replication forks relied on conventional immunofluorescence and immunoprecipitation assays of 5'-ethylene-2'-deoxyuridine (EdU) labeled DNA (iPOND). While greatly informative and important, these methods lack specificities for nascent fork interactions (, IF) or assay an average change of millions of cells without single-cell resolution (, iPOND). The assay system described here combines proximity ligation assay (PLA) with EdU coupled click-iT chemistry, which we termed " protein interaction with nascent DNA replication forks (SIRF)". This method enables sensitive and quantitative analysis of protein interactions with nascent DNA replication forks with single-cell resolution, and can further be paired with conventional immunofluorescence marker analysis for added multi-parameter analysis.
DNA复制是遗传信息从亲代细胞传递到子代细胞所必需的一个基本过程。异常的DNA复制过程与多种疾病表型相关，包括发育缺陷、衰老紊乱、范可尼贫血等血液疾病、炎症增加以及癌症。因此，开发用于研究与无差错DNA复制过程相关蛋白质的工具至关重要。到目前为止，研究与新生复制叉相关蛋白质的方法依赖于对5'-乙烯-2'-脱氧尿苷（EdU）标记DNA的传统免疫荧光和免疫沉淀分析（iPOND）。尽管这些方法提供了大量信息且很重要，但它们缺乏对新生叉相互作用（IF）的特异性，或者在没有单细胞分辨率的情况下分析数百万个细胞的平均变化（iPOND）。本文描述的检测系统将邻近连接分析（PLA）与EdU偶联的点击化学相结合，我们将其称为“蛋白质与新生DNA复制叉相互作用（SIRF）”。该方法能够以单细胞分辨率对蛋白质与新生DNA复制叉的相互作用进行灵敏且定量的分析，并且可以进一步与传统免疫荧光标记分析相结合，以进行额外的多参数分析。
