生物谷報道:聯(lián)會現(xiàn)象是細(xì)胞在分裂過程中遺傳物質(zhì)復(fù)制前的一個過程。位點特異的重組反應(yīng)發(fā)生于復(fù)雜的聯(lián)會復(fù)合體中,,反應(yīng)過程中缺乏高能量的輔助因子,,是等能的化學(xué)反應(yīng),該反應(yīng)系統(tǒng)依賴于對重組的方向和拓?fù)浣Y(jié)構(gòu)的調(diào)節(jié),。由于缺乏調(diào)節(jié)組件的結(jié)構(gòu)信息,,目前對聯(lián)會復(fù)合體的結(jié)構(gòu)以及位點特異重組反應(yīng)的分子基礎(chǔ)還了解甚少。
解離酶Sin是絲氨酸重組酶家族的一員,,由金黃色葡萄球菌中的多抗性質(zhì)粒編碼,。它能夠裝配復(fù)雜的聯(lián)會復(fù)合體,利用其中的負(fù)超螺旋結(jié)構(gòu)促進(jìn)定向正確的位點間重組反應(yīng),。在此聯(lián)會復(fù)合體中,,DNA結(jié)合于Sin二聚體的位點Ⅰ和Ⅱ。為了解此聯(lián)會復(fù)合體的整體結(jié)構(gòu)和其中的相互調(diào)節(jié)和催化過程,,來自美國和英國的Mouw等科學(xué)家確定了位點Ⅱ結(jié)合DNA雙螺旋的Sin二聚體晶體結(jié)構(gòu),。結(jié)果發(fā)現(xiàn)每個Sin單體由N末端催化結(jié)構(gòu)域和C末端的α螺旋-轉(zhuǎn)角-α螺旋結(jié)構(gòu)域組成。Sin可能通過形成穩(wěn)定的四聚體起作用,,而其中二聚體間的一個重要界面是C末端結(jié)構(gòu)域,,突變體分析和模型模擬均很好地支持了這一結(jié)構(gòu)推斷。通過對突變體的分析,,研究人員發(fā)現(xiàn)C末端結(jié)構(gòu)域是發(fā)生在位點Ⅱ的聯(lián)會所必須的,,同時也是激發(fā)位點Ⅰ的重組反應(yīng)所必須的,,該激活作用依賴于N末端催化結(jié)構(gòu)域中R54殘基的存在。該研究表明Sin的催化作用受到輔助位點的調(diào)節(jié)作用,,能夠使聯(lián)會復(fù)合體更精確定向,,防止發(fā)生錯誤的DNA雙鏈斷裂和重組。而目前對真核生物的基因組操作中所用的位點特異連接酶可以同時催化正向和反向的連接,,與底物的拓?fù)浣Y(jié)構(gòu)無關(guān),。該研究結(jié)果將促進(jìn)開發(fā)更有效的的連接酶工具用于基因工程研究。(生物谷www.bioon.com)
生物谷推薦原始出處:
Molecular Cell,,Vol 30, 145-155, 25 April 2008,,Kent W. Mouw, Phoebe A. Rice
Architecture of a Serine Recombinase-DNA Regulatory Complex
Kent W. Mouw,1 Sally-J. Rowland,2 Mark M. Gajjar,1,3 Martin R. Boocock,2 W. Marshall Stark,2 and Phoebe A. Rice1,
1 Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA
2 Division of Molecular Genetics, FBLS, University of Glasgow, Glasgow G12 8QQ, Scotland, UK
Summary
An essential feature of many site-specific recombination systems is their ability to regulate the direction and topology of recombination. Resolvases from the serine recombinase family assemble an interwound synaptic complex that harnesses negative supercoiling to drive the forward reaction and promote recombination between properly oriented sites. To better understand the interplay of catalytic and regulatory functions within these synaptic complexes, we have solved the structure of the regulatory site synapse in the Sin resolvase system. It reveals an unexpected synaptic interface between helix-turn-helix DNA-binding domains that is also highlighted in a screen for synapsis mutants. The tetramer defined by this interface provides the foundation for a robust model of the synaptic complex, assembled entirely from available crystal structures, that gives insight into how the catalytic activity of Sin and other serine recombinases may be regulated.