一种用于测量 DNA 复制速率的活细胞成像方法。

A Live-Cell Imaging Approach for Measuring DNA Replication Rates.

机构信息

Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be'er Sheva 8410501, Israel.

出版信息

Cell Rep. 2018 Jul 3;24(1):252-258. doi: 10.1016/j.celrep.2018.06.018.

DOI:10.1016/j.celrep.2018.06.018

PMID:29972785

Abstract

We describe a simple and direct approach to measure the progression of single DNA replication forks in living cells by monitoring two fluorescently labeled loci downstream of an origin of replication. We employ this approach to investigate the roles of several leading and lagging strand factors in overall replisome function and show that fork progression is strongly dependent on proper maturation of Okazaki fragments. We also demonstrate how related cellular phenotypes, such as cell-cycle progression and the dynamics of sister chromatid cohesion, may be simultaneously monitored and correlated to DNA replication at the single-cell level.

摘要

我们描述了一种简单直接的方法，通过监测复制起始点下游的两个荧光标记位点，来测量活细胞中单 DNA 复制叉的进展。我们利用这种方法研究了几个前导链和滞后链因子在整体复制体功能中的作用，并表明叉的进展强烈依赖于 Okazaki 片段的适当成熟。我们还展示了如何同时监测相关的细胞表型，如细胞周期进程和姐妹染色单体凝聚的动态，并将其与单细胞水平的 DNA 复制相关联。