Key Laboratory of Environment and Female Reproductive Health, West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu, China.
Institute for Chemical Carcinogenesis, Guangzhou Medical University, Xinzao, Panyu District, Guangzhou, China.
Mutagenesis. 2019 Dec 19;34(4):355-361. doi: 10.1093/mutage/gez013.
Abasic site as a common DNA lesion blocks DNA replication and is highly mutagenic. Protein interactions in T7 DNA replisome facilitate DNA replication and translesion DNA synthesis. However, bypass of an abasic site by T7 DNA replisome has never been investigated. In this work, we used T7 DNA replisome and T7 DNA polymerase alone as two models to study DNA replication on encountering an abasic site. Relative to unmodified DNA, abasic site strongly inhibited primer extension and completely blocked strand-displacement DNA synthesis, due to the decreased fraction of enzyme-DNA productive complex and the reduced average extension rates. Moreover, abasic site at DNA fork inhibited the binding of DNA polymerase or helicase onto fork and the binding between polymerase and helicase at fork. Notably and unexpectedly, we found DNA polymerase alone bypassed an abasic site on primer/template (P/T) substrate more efficiently than did polymerase and helicase complex bypass it at fork. The presence of gp2.5 further inhibited the abasic site bypass at DNA fork. Kinetic analysis showed that this inhibition at fork relative to that on P/T was due to the decreased fraction of productive complex instead of the average extension rates. Therefore, we found that protein interactions in T7 DNA replisome inhibited the bypass of DNA lesion, different from all the traditional concept that protein interactions or accessory proteins always promote DNA replication and DNA damage bypass, providing new insights in translesion DNA synthesis performed by DNA replisome.
碱基缺失位点是一种常见的 DNA 损伤，它会阻止 DNA 复制并具有高度诱变作用。T7 DNA 复制酶中的蛋白相互作用有助于 DNA 复制和跨损伤 DNA 合成。然而，T7 DNA 复制酶是否能绕过碱基缺失位点尚未得到研究。在这项工作中，我们使用 T7 DNA 复制酶和 T7 DNA 聚合酶本身作为两种模型，研究在遇到碱基缺失位点时 DNA 的复制情况。与未修饰的 DNA 相比，碱基缺失位点强烈抑制引物延伸，完全阻断链置换 DNA 合成，这是由于酶-DNA 有效复合物的比例降低，以及平均延伸速率降低。此外，碱基缺失位点在 DNA 叉处抑制了 DNA 聚合酶或解旋酶与叉的结合，以及聚合酶和解旋酶在叉处的结合。值得注意的是，令人惊讶的是，我们发现 DNA 聚合酶本身比聚合酶和解旋酶复合物在叉处更有效地绕过引物/模板（P/T）底物上的碱基缺失位点。gp2.5 的存在进一步抑制了 DNA 叉处碱基缺失的绕过。动力学分析表明，相对于 P/T 处的抑制，这种在叉处的抑制是由于有效复合物的比例降低，而不是平均延伸速率降低。因此，我们发现 T7 DNA 复制酶中的蛋白相互作用抑制了 DNA 损伤的绕过，这与蛋白相互作用或辅助蛋白总是促进 DNA 复制和 DNA 损伤绕过的传统观念不同，为 DNA 复制酶进行的跨损伤 DNA 合成提供了新的见解。
