Physical and functional mapping of the replication protein A interaction domain of the Werner and Bloom syndrome helicases
Physical and functional mapping of the replication protein A interaction domain of the Werner and Bloom syndrome helicases
复制标题
DOI：
10.1074/jbc.m500653200
复制
发表时间：
2005-08-19
影响因子：
4.8
通讯作者：
Brosh, RM
中科院分区：
文献类型：
--
作者：
Doherty, KM;Sommers, JA;Brosh, RM
单链DNA结合蛋白复制蛋白A(RPA)与几种人类RecQ DNA解旋酶相互作用，这些解旋酶在维持基因组稳定性方面具有重要作用；然而，RPA刺激DNA解离的机制尚不清楚。为了定位Werner综合征解旋酶(WRN)与RPA相互作用的区域，我们进行了酵母双杂交研究、WRN亲和下拉实验和纯化的重组WRN蛋白片段的酶联免疫吸附试验。结果表明，WRN有两个RPA结合位点，一个是高亲和力的N-末端，一个是低亲和力的C-末端。根据作图研究的结果，我们试图确定包含高亲和力RPA相互作用位点的WRN N-末端区域是否对RPA刺激WRN解旋酶活性起重要作用。为了做到这一点，我们测试了一个具有催化活性的WRN解旋酶结构域片段(WRNH-R)，它缺乏N-末端RPA相互作用位点，因为它有能力解离长DNA双链底物，而野生型酶只有在RPA存在的情况下才能有效地解离。与全长WRN相比，WRNH-R解旋酶活性在依赖RPA的部分双链底物上显著降低，尽管存在RPA。这些结果清楚地表明，尽管WRNH-R在短双链底物上具有与全长WRN类似的解旋酶活性，但其解离依赖于RPA的WRN解旋酶底物的能力显著减弱。同样，缺乏N-末端RPA相互作用位点的Bloom综合征解旋酶(BLM)结构域片段BLM642-1290也解开了类似于野生型BLM的短DNA双链底物，但其解离长DNA底物的能力严重受损，而全长BLM解旋酶可以在RPA存在下解离。这些结果表明，RPA与WRN或BLM解旋酶之间的物理相互作用在RPA刺激解旋酶催化的DNA解离的机制中起着重要作用。
The single-stranded DNA-binding protein replication protein A (RPA) interacts with several human RecQ DNA helicases that have important roles in maintaining genomic stability; however, the mechanism for RPA stimulation of DNA unwinding is not well understood. To map regions of Werner syndrome helicase (WRN) that interact with RPA, yeast two-hybrid studies, WRN affinity pull-down experiments and enzyme-linked immunosorbent assays with purified recombinant WRN protein fragments were performed. The results indicated that WRN has two RPA binding sites, a high affinity N-terminal site, and a lower affinity C-terminal site. Based on results from mapping studies, we sought to determine if the WRN N-terminal region harboring the high affinity RPA interaction site was important for RPA stimulation of WRN helicase activity. To accomplish this, we tested a catalytically active WRN helicase domain fragment (WRNH-R) that lacked the N-terminal RPA interaction site for its ability to unwind long DNA duplex substrates, which the wild-type enzyme can efficiently unwind only in the presence of RPA. WRNH-R helicase activity was significantly reduced on RPA-dependent partial duplex substrates compared with full-length WRN despite the presence of RPA. These results clearly demonstrate that, although WRNH-R had comparable helicase activity to full-length WRN on short duplex substrates, its ability to unwind RPA-dependent WRN helicase substrates was significantly impaired. Similarly, a Bloom syndrome helicase (BLM) domain fragment, BLM642-1290, that lacked its N-terminal RPA interaction site also unwound short DNA duplex substrates similar to wild-type BLM, but was severely compromised in its ability to unwind long DNA substrates that full-length BLM helicase could unwind in the presence of RPA. These results suggest that the physical interaction between RPA and WRN or BLM helicases plays an important role in the mechanism for RPA stimulation of helicase-catalyzed DNA unwinding.