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.

中文翻译：

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T7 DNA复制体中的蛋白质相互作用可抑制DNA聚合酶旁路无碱基位点。

无碱基位点是常见的DNA损伤，会阻止DNA复制，并具有高度致突变性。T7 DNA复制体中的蛋白质相互作用促进DNA复制和跨病变DNA合成。然而，从未研究过T7 DNA复制体绕过无碱基位点。在这项工作中，我们仅使用T7 DNA复制体和T7 DNA聚合酶作为两个模型来研究遇到无碱基位点时的DNA复制。相对于未修饰的DNA，由于酶-DNA生产复合物的分数降低和平均延伸率降低，无碱基位点强烈抑制了引物延伸并完全阻断了链置换DNA的合成。而且，DNA叉处的无碱基位点抑制了DNA聚合酶或解旋酶在叉上的结合以及聚合酶与解旋酶在叉处的结合。出乎意料的是，我们发现，与聚合酶和解旋酶复合物绕叉时相比，单独使用DNA聚合酶更有效地绕过引物/模板（P / T）底物上的无碱基位点。gp2.5的存在进一步抑制了DNA叉处的无碱基位点旁路。动力学分析表明，相对于P / T而言，这种对叉的抑制作用是由于生产复合物的分数降低而不是平均延伸率所致。因此，我们发现T7 DNA复制体中的蛋白质相互作用抑制了DNA损伤的旁路，这不同于所有传统观念，即蛋白质相互作用或辅助蛋白总是促进DNA复制和DNA损伤旁路，从而为DNA复制体进行的跨病变DNA合成提供了新的见解。 。