Doherty Kevin M, Sommers Joshua A, Gray Matthew D, Lee Jae Wan, von Kobbe Cayetano, Thoma Nicolas H, Kureekattil Raichal P, Kenny Mark K, Brosh Robert M
Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Baltimore, Maryland 21224, USA.
J Biol Chem. 2005 Aug 19;280(33):29494-505. doi: 10.1074/jbc.M500653200. Epub 2005 Jun 17.
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 (WRN(H-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. WRN(H-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 WRN(H-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, BLM(642-1290), 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.
单链DNA结合蛋白复制蛋白A（RPA）与几种在维持基因组稳定性中起重要作用的人类RecQ DNA解旋酶相互作用；然而，RPA刺激DNA解旋的机制尚未完全清楚。为了绘制与RPA相互作用的沃纳综合征解旋酶（WRN）区域，进行了酵母双杂交研究、WRN亲和下拉实验以及使用纯化的重组WRN蛋白片段的酶联免疫吸附测定。结果表明，WRN有两个RPA结合位点，一个高亲和力的N端位点和一个低亲和力的C端位点。基于定位研究的结果，我们试图确定含有高亲和力RPA相互作用位点的WRN N端区域对于RPA刺激WRN解旋酶活性是否重要。为了实现这一点，我们测试了一个缺乏N端RPA相互作用位点的具有催化活性的WRN解旋酶结构域片段（WRN(H-R)），看它解开长DNA双链底物的能力，而野生型酶只有在RPA存在时才能有效地解开这种底物。尽管存在RPA，但与全长WRN相比，WRN(H-R)解旋酶活性在RPA依赖的部分双链底物上显著降低。这些结果清楚地表明，虽然WRN(H-R)在短双链底物上具有与全长WRN相当的解旋酶活性，但其解开RPA依赖的WRN解旋酶底物的能力明显受损。同样，一个缺乏其N端RPA相互作用位点的布卢姆综合征解旋酶（BLM）结构域片段BLM(642 - 1290)，也能解开类似于野生型BLM的短DNA双链底物，但在解开全长BLM解旋酶在RPA存在时能解开的长DNA底物的能力上严重受损。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解旋机制中起重要作用。
