Overlapping roles for PARP1 and PARP2 in the recruitment of endogenous XRCC1 and PNKP into oxidized chromatin.
Overlapping roles for PARP1 and PARP2 in the recruitment of endogenous XRCC1 and PNKP into oxidized chromatin.
复制标题
DOI：
10.1093/nar/gkw1246
复制
发表时间：
2017-03-17
影响因子：
14.9
通讯作者：
Caldecott KW
中科院分区：
文献类型：
--
作者：
Hanzlikova H;Gittens W;Krejcikova K;Zeng Z;Caldecott KW
DNA单链断裂修复的关键步骤是支架蛋白XRCC 1的快速募集，该蛋白与修复过程的多种酶组分相互作用，稳定和刺激。XRCC 1的募集由PARP 1促进，PARP 1是一种在DNA损伤后被激活并合成XRCC 1直接结合的ADP-核糖聚合物的酶。然而，细胞具有另外两种DNA链断裂诱导的PARP酶，PARP 2和PARP 3，其作用尚不清楚。为了解决它们参与将内源性XRCC 1招募到氧化染色质中的问题，我们建立了“同基因”人二倍体细胞，其中PARP 1和/或PARP 2或PARP 3缺失。令人惊讶的是，我们表明PARP 1或PARP 2足以在氧化性单链断裂（SSB）处进行接近正常的XRCC 1募集，如在H2 O2处理后需要损失两种蛋白质以大大减少或消除XRCC 1染色质结合所指示的。对于PNKP观察到类似的结果; XRCC 1蛋白伴侣对氧化SSB的修复很重要。值得注意的是，在H2 O2处理后，需要高于IC 50>1000倍的PARP抑制剂浓度来消除ADP-核糖基化和XRCC 1染色质结合。这些结果表明，由PARP 1或PARP 2合成的非常低水平的ADP-核糖基化足以在氧化应激后进行XRCC 1募集。
A critical step of DNA single-strand break repair is the rapid recruitment of the scaffold protein XRCC1 that interacts with, stabilizes and stimulates multiple enzymatic components of the repair process. XRCC1 recruitment is promoted by PARP1, an enzyme that is activated following DNA damage and synthesizes ADP-ribose polymers that XRCC1 binds directly. However, cells possess two other DNA strand break-induced PARP enzymes, PARP2 and PARP3, for which the roles are unclear. To address their involvement in the recruitment of endogenous XRCC1 into oxidized chromatin we have established ‘isogenic’ human diploid cells in which PARP1 and/or PARP2, or PARP3 are deleted. Surprisingly, we show that either PARP1 or PARP2 are sufficient for near-normal XRCC1 recruitment at oxidative single-strand breaks (SSBs) as indicated by the requirement for loss of both proteins to greatly reduce or ablate XRCC1 chromatin binding following H2O2 treatment. Similar results were observed for PNKP; an XRCC1 protein partner important for repair of oxidative SSBs. Notably, concentrations of PARP inhibitor >1000-fold higher than the IC50 were required to ablate both ADP-ribosylation and XRCC1 chromatin binding following H2O2 treatment. These results demonstrate that very low levels of ADP-ribosylation, synthesized by either PARP1 or PARP2, are sufficient for XRCC1 recruitment following oxidative stress.