Mechanistic insight into AP-endonuclease 1 cleavage of abasic sites at stalled replication fork mimics.
Mechanistic insight into AP-endonuclease 1 cleavage of abasic sites at stalled replication fork mimics.
复制标题
DOI：
10.1093/nar/gkad481
复制
发表时间：
2023-07-21
影响因子：
14.9
通讯作者：
中科院分区：
文献类型：
--
作者：
许多类型的损伤，包括脱碱基位点，阻断复制DNA聚合酶，导致复制叉解偶联和产生ssDNA。AP-内切核酸酶1（APE 1）已显示切割ssDNA中的脱碱基位点。重要的是，APE 1在复制叉处切割ssDNA具有重要的生物学意义，因为它会产生双链断裂，从而使复制叉崩溃。尽管如此，APE 1在复制叉处加工脱碱基位点的分子基础和效率仍然难以捉摸。在这里，我们研究APE 1切割的无碱基底物，模仿APE 1的相互作用在停滞的复制叉或差距。我们确定APE 1对这些底物（如dsDNA）具有稳健的活性，并报告了切割和产物释放的速率。X射线结构可视化APE 1活性位点，突出显示了类似的机制用于处理ssDNA底物，作为dsDNA上的典型APE 1活性。然而，突变分析显示R177对于APE 1 ssDNA切割机制是唯一关键的。此外，我们研究了APE 1和复制蛋白A（RPA）之间的相互作用，RPA是复制叉处的主要ssDNA结合蛋白，揭示了APE 1可以切割脱碱基位点，而RPA仍然与DNA结合。总之，这项工作提供了分子水平的见解，通过APE 1的无碱基ssDNA加工，包括RPA的存在。
Many types of damage, including abasic sites, block replicative DNA polymerases causing replication fork uncoupling and generating ssDNA. AP-Endonuclease 1 (APE1) has been shown to cleave abasic sites in ssDNA. Importantly, APE1 cleavage of ssDNA at a replication fork has significant biological implications by generating double strand breaks that could collapse the replication fork. Despite this, the molecular basis and efficiency of APE1 processing abasic sites at replication forks remain elusive. Here, we investigate APE1 cleavage of abasic substrates that mimic APE1 interactions at stalled replication forks or gaps. We determine that APE1 has robust activity on these substrates, like dsDNA, and report rates for cleavage and product release. X-ray structures visualize the APE1 active site, highlighting an analogous mechanism is used to process ssDNA substrates as canonical APE1 activity on dsDNA. However, mutational analysis reveals R177 to be uniquely critical for the APE1 ssDNA cleavage mechanism. Additionally, we investigate the interplay between APE1 and Replication Protein A (RPA), the major ssDNA-binding protein at replication forks, revealing that APE1 can cleave an abasic site while RPA is still bound to the DNA. Together, this work provides molecular level insights into abasic ssDNA processing by APE1, including the presence of RPA.
登录
查看更多内容
影响因子：
3.7
作者：
Gibb B;Ye LF;Gergoudis SC;Kwon Y;Niu H;Sung P;Greene EC
通讯作者：
Greene EC
影响因子：
16.8
作者：
Freudenthal BD;Beard WA;Cuneo MJ;Dyrkheeva NS;Wilson SH
通讯作者：
Wilson SH
影响因子：
2.9
作者：
He, Hongzhen;Chen, Qujia;Georgiadis, Millie M.
通讯作者：
Georgiadis, Millie M.
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
5.6
作者：
Berquist, Brian R.;McNeill, Daniel R.;Wilson, David M., III
通讯作者：
Wilson, David M., III