Genschel Jochen, Bazemore Laura R, Modrich Paul
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 2002 Apr 12;277(15):13302-11. doi: 10.1074/jbc.M111854200. Epub 2002 Jan 24.
We have partially purified a human activity that restores mismatch-dependent, bi-directional excision to a human nuclear extract fraction depleted for one or more mismatch repair excision activities. Human EXOI co-purifies with the excision activity, and the purified activity can be replaced by near homogeneous recombinant hEXOI. Despite the reported 5' to 3' hydrolytic polarity of this activity, hEXOI participates in mismatch-provoked excision directed by a strand break located either 5' or 3' to the mispair. When the strand break that directs repair is located 3' to the mispair, hEXOI- and mismatch-dependent gap formation in excision-depleted extracts requires both hMutSalpha and hMutLalpha. However, excision directed by a 5' strand break requires hMutSalpha but can occur in absence of hMutLalpha. In systems comprised of pure components, the 5' to 3' hydrolytic activity of hEXOI is activated by hMutSalpha in a mismatch-dependent manner. These observations indicate a hydrolytic function for hEXOI in 5'-heteroduplex correction. The involvement of hEXOI in 3'-heteroduplex repair suggests that it has a regulatory/structural role in assembly of the 3'-excision complex or that the protein possesses a cryptic 3' to 5' hydrolytic activity.
我们已经部分纯化了一种人类活性物质，该物质可将错配依赖性双向切除恢复到一种人类核提取物组分中，该组分缺乏一种或多种错配修复切除活性。人EXOI与切除活性共纯化，并且纯化的活性可以被近乎纯的重组hEXOI替代。尽管报道了该活性具有5'至3'的水解极性，但hEXOI参与由位于错配5'或3'端的链断裂引发的错配诱导切除。当指导修复的链断裂位于错配的3'端时，hEXOI和切除缺陷提取物中错配依赖性缺口形成需要hMutSalpha和hMutLalpha两者。然而，由5'链断裂指导的切除需要hMutSalpha，但可以在没有hMutLalpha的情况下发生。在由纯组分组成的系统中，hEXOI的5'至3'水解活性以错配依赖性方式被hMutSalpha激活。这些观察结果表明hEXOI在5'-异源双链体校正中具有水解功能。hEXOI参与3'-异源双链体修复表明它在3'-切除复合物的组装中具有调节/结构作用，或者该蛋白质具有隐蔽的3'至5'水解活性。
