Symmetric activity of DNA polymerases at and recruitment of exonuclease ExoR and of PolA to the Bacillus subtilis replication forks
Symmetric activity of DNA polymerases at and recruitment of exonuclease ExoR and of PolA to the Bacillus subtilis replication forks
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DOI：
10.1093/nar/gkz554
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发表时间：
2019-09-19
影响因子：
14.9
通讯作者：
Graumann, Peter L.
中科院分区：
文献类型：
--
作者：
Hernandez-Tamayo, Rogelio;Oviedo-Bocanegra, Luis M.;Graumann, Peter L.
DNA复制叉本质上是不对称的，在细胞周期中遇到DNA修饰时可能会停止。我们在单分子水平上研究了枯草芽孢杆菌中三种DNA聚合酶和一种核酸外切酶的真实的时间动力学。PolC和DnaE以对称的方式工作，并显示出相似的停留时间。在添加DNA损伤后，它们的静态分数和停留时间减少，与复制叉的重建增加一致。只有一小部分的复制叉表现出活性聚合酶的损失，这表明DNA修复过程中相对稳健的活性。相反，PolA，同源物的聚合酶I和核酸外切酶ExoR很少出现在叉子在不受干扰的复制，但被招募到复制叉诱导DNA损伤后。PolA或ExoR的蛋白质动力学在指数生长和DNA修复过程中彼此不存在的情况下发生改变，表明功能重叠。纯化的ExoR显示核酸外切酶活性，并在体外优先结合具有5'突出端的DNA。我们的分析支持的想法，两个复制DNA聚合酶一起工作的滞后链，而只有PolC的行为在前导链，PolA和ExoR在DNA修复过程中的复制叉进行诱导功能。
DNA replication forks are intrinsically asymmetric and may arrest during the cell cycle upon encountering modifications in the DNA. We have studied real time dynamics of three DNA polymerases and an exonuclease at a single molecule level in the bacterium Bacillus subtilis. PolC and DnaE work in a symmetric manner and show similar dwell times. After addition of DNA damage, their static fractions and dwell times decreased, in agreement with increased re-establishment of replication forks. Only a minor fraction of replication forks showed a loss of active polymerases, indicating relatively robust activity during DNA repair. Conversely, PolA, homolog of polymerase I and exonuclease ExoR were rarely present at forks during unperturbed replication but were recruited to replications forks after induction of DNA damage. Protein dynamics of PolA or ExoR were altered in the absence of each other during exponential growth and during DNA repair, indicating overlapping functions. Purified ExoR displayed exonuclease activity and preferentially bound to DNA having 5' overhangs in vitro. Our analyses support the idea that two replicative DNA polymerases work together at the lagging strand whilst only PolC acts at the leading strand, and that PolA and ExoR perform inducible functions at replication forks during DNA repair.