科研人员报告了一种在活细菌中观察DNA修复的技术。
诱变剂导致的DNA核苷酸碱基的损伤被一个称为切补修复的过程所逆转，这个过程是由一组酶执行的。其中两个酶——取代失踪的核苷酸的DNA聚合酶I与把DNA链连接在一起从而帮助弥合切口的DNA连接酶——在从细菌到人的一系列生物中执行了这个修复过程的最终步骤，但是迄今为止这些酶发挥作用的过程还尚未被直接观察到。
Stephan Uphoff及其同事使用一种称为光敏定位显微镜的技术把这两种酶在活的大肠杆菌中的单个分子的活动显现出来。这组作者把单个酶分子与DNA结合的痕迹解释为DNA合成与连接的证据，他们发现DNA修复点遍布于整个细胞之中。这组作者还发现，对于聚合酶，单个修复事件持续了2.1秒，而对于连接酶，单个修复时间持续了2.5秒；在实验诱发DNA损伤的数分钟内，这两种酶的活动增加到了基线状态的5倍。
在DNA没有损伤的一个细菌细胞内，在任何时间里，在大约400个聚合酶的拷贝中只有2.7%的拷贝处于复制和修复的活动当中，而在大约200个连接酶的拷贝中大约有3.8%的拷贝处于复制和修复的活动当中。这组作者发现当细胞受到DNA损伤的时候，这两种酶都把它们寿命的80%以上用于寻找有毒的中间物以进行修复工作，因此最小化了DNA缺口与切口的生存时间。
这组作者说，直接测量活细胞的DNA修复率可能会引出一些定量模型，它们能够帮助科研人员预测生物如何能够对DNA的损伤做出响应。（生物谷Bioon.com）
doi:10.1073/pnas.1301804110
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Single-molecule DNA repair in live bacteria
Stephan Uphoff, Rodrigo Reyes-Lamothe, Federico Garza de Leon, David J. Sherratt, and Achillefs N. Kapanidis
Cellular DNA damage is reversed by balanced repair pathways that avoid accumulation of toxic intermediates. Despite their importance, the organization of DNA repair pathways and the function of repair enzymes in vivo have remained unclear because of the inability to directly observe individual reactions in living cells. Here, we used photoactivation, localization, and tracking in live Escherichia coli to directly visualize single fluorescent labeled DNA polymerase I (Pol) and ligase (Lig) molecules searching for DNA gaps and nicks, performing transient reactions, and releasing their products. Our general approach provides enzymatic rates and copy numbers, substrate-search times, diffusion characteristics, and the spatial distribution of reaction sites, at the single-cell level, all in one measurement. Single repair events last 2.1 s (Pol) and 2.5 s (Lig), respectively. Pol and Lig activities increased fivefold over the basal level within minutes of DNA methylation damage; their rates were limited by upstream base excision repair pathway steps. Pol and Lig spent >80% of their time searching for free substrates, thereby minimizing both the number and lifetime of toxic repair intermediates. We integrated these single-molecule observations to generate a quantitative, systems-level description of a model repair pathway in vivo.
