生物谷報(bào)道：臺(tái)灣中研院生化所的王惠鈞（Andrew H.-J.Wang）和王廷方(Ting-Fang Wang)博士領(lǐng)導(dǎo)的一個(gè)研究組發(fā)現(xiàn)RecA家族重組酶功能是充當(dāng)DNA損傷修復(fù)的一種新型的回轉(zhuǎn)馬達(dá)蛋白質(zhì),。研究組近期發(fā)表了兩篇有關(guān)RecA家族重組酶的結(jié)構(gòu)生物學(xué)相關(guān)文章,。一篇文章發(fā)表9月12日的網(wǎng)絡(luò)版雜志PLoS ONE上，另外一篇發(fā)表在今年2月28日的Nucleic Acids Reseach雜志上,。
同源重組（HR）是精確修復(fù)受損DNA的一種機(jī)制,，該過(guò)程利用來(lái)自同伴DNA的同源序列作為模板。這個(gè)過(guò)程包括將兩個(gè)DNA分子集合在一起,，搜索同源序列和交換DNA鏈,。
RecA家族蛋白是同源重組的中心重組酶。這個(gè)家族的成員包括原核RecA,、古細(xì)菌RadA和真核Rad51和Dmc1,。它們?cè)诩?xì)胞增殖、基因組維護(hù)和遺傳多樣性中起到重要作用,，尤其是在高等真核生物中,。例如，缺少Rad51的脊椎動(dòng)物細(xì)胞會(huì)累積染色體缺口,。Rad51和它的減數(shù)分裂特異性同系物Dmc1也是減數(shù)分裂過(guò)程必不可少的酶,。
自從研究人員發(fā)現(xiàn)RecA家族蛋白，研究人員就推測(cè)RecA和它的類(lèi)似物只形成61右手螺旋細(xì)絲（每個(gè)螺旋由六個(gè)蛋白單體組成）,，然后水解ATP來(lái)促進(jìn)同源重組和重組性的DNA修復(fù),。然而始終存在一個(gè)爭(zhēng)議性的謎團(tuán)——在鏈交換反應(yīng)過(guò)程中，ATP能量如何促進(jìn)DNA的旋轉(zhuǎn),。
通過(guò)使用X射線晶體衍射和原子顯微鏡技術(shù),，王博士的研究組給出了這個(gè)問(wèn)題的答案。他們報(bào)道說(shuō),，古細(xì)菌Sulfolobus solfataricus的RadA蛋白還能夠自我聚合成一種31右手螺旋細(xì)絲（每個(gè)螺旋有3個(gè)單體）和一種43右手螺旋細(xì)絲（每個(gè)螺旋有4個(gè)單體）,。
另外的生物物理和生物化學(xué)分析揭示出RecA家族蛋白可能將ATP結(jié)合和水解過(guò)程以一種促進(jìn)核蛋白順時(shí)針旋轉(zhuǎn)的方式來(lái)耦聯(lián)到DNA鏈的交換過(guò)程中。尤其是61RadA螺旋細(xì)絲順時(shí)針旋轉(zhuǎn)變成31延伸的右手螺旋細(xì)絲,，然后轉(zhuǎn)化成43左手螺旋纖絲,。因此,，RadA蛋白中的所有DNA結(jié)合motifs一致運(yùn)動(dòng)來(lái)介導(dǎo)DNA結(jié)合、同源配對(duì)和鏈交換,。因此,，ATP能量不僅被用于DNA底物，而且還被RecA家族蛋白纖絲所利用,。這種新的模型挑戰(zhàn)了所有目前提出的假說(shuō),。
王惠鈞是臺(tái)灣大學(xué)化學(xué)系所畢業(yè)，美國(guó)伊利諾大學(xué)香檳分?；瘜W(xué)博士,、麻省理工學(xué)院生物系博士后研究員到首席與資深研究員，伊大香檳分校細(xì)胞及結(jié)構(gòu)生物學(xué)系教授,，中研院分子生物研究所學(xué)術(shù)咨詢(xún)委員,，歐洲生物化學(xué)學(xué)報(bào)編者，臺(tái)大生化科學(xué)所教授,。
2000年當(dāng)選中央研究院院士后,，回臺(tái)灣任中研院生物化學(xué)研究所特聘研究員兼所長(zhǎng)至今，王惠鈞又陸續(xù)接任中華民國(guó)生物物理學(xué)會(huì)理事長(zhǎng),、臺(tái)灣生物化學(xué)及分子生物學(xué)會(huì)理事長(zhǎng),、臺(tái)灣蛋白質(zhì)體學(xué)會(huì)理事長(zhǎng)、世界蛋白質(zhì)體學(xué)會(huì)（HUPO）理事長(zhǎng),、亞太蛋白質(zhì)體學(xué)會(huì)（AOHUPO）理事,；他還曾獲美國(guó)生化及分生學(xué)會(huì)、美國(guó)化學(xué)研究所,、美國(guó)科學(xué)促進(jìn)協(xié)會(huì)等的名譽(yù)會(huì)員殊榮,。
王惠鈞一直致力提升臺(tái)灣的生化學(xué)術(shù)水平，運(yùn)用他生物結(jié)構(gòu)學(xué)研究專(zhuān)長(zhǎng),，展開(kāi)生物結(jié)構(gòu)學(xué)與功能基因體學(xué)的探討,，以了解重要的生物系統(tǒng)功能，主要技術(shù)平臺(tái)有高效能的蛋白質(zhì)同步輻射結(jié)晶學(xué)與蛋白質(zhì)體學(xué),，其它先進(jìn)技術(shù)也可在需要時(shí)加入使用,。
目前他正在努力四項(xiàng)領(lǐng)域，結(jié)構(gòu)酶學(xué)是研究幾個(gè)具藥物發(fā)展?jié)摿Φ拿溉?，如蛋白質(zhì)分解酶,、醣化酶、磷酸水解酶,、prenyltransferases,；蛋白質(zhì)-DNA與抗癌藥物-DNA交互作用是研究DNA結(jié)合蛋白和重要的抗癌藥物與DNA的交互作用。
原始出處:
PLoS one
Received: July 20, 2007; Accepted: August 16, 2007; Published: September 12, 2007
Structural and Functional Analyses of Five Conserved Positively Charged Residues in the L1 and N-Terminal DNA Binding Motifs of Archaeal RadA Protein
Li-Tzu Chen1,3, Tzu-Ping Ko3, Yu-Wei Chang1,3, Kuei-An Lin3, Andrew H.-J. Wang1,2,3,4*, Ting-Fang Wang1,3*
1 Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan, 2 Department of Life Sciences, National Taiwan University, Taipei, Taiwan, 3 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, 4 National Core Facility of High-Throughput Protein Crystallography, Academia Sinica, Taipei, Taiwan
RecA family proteins engage in an ATP-dependent DNA strand exchange reaction that includes a ssDNA nucleoprotein helical filament and a homologous dsDNA sequence. In spite of more than 20 years of efforts, the molecular mechanism of homology pairing and strand exchange is still not fully understood. Here we report a crystal structure of Sulfolobus solfataricus RadA overwound right-handed filament with three monomers per helical pitch. This structure reveals conformational details of the first ssDNA binding disordered loop (denoted L1 motif) and the dsDNA binding N-terminal domain (NTD). L1 and NTD together form an outwardly open palm structure on the outer surface of the helical filament. Inside this palm structure, five conserved basic amino acid residues (K27, K60, R117, R223 and R229) surround a 25 Å pocket that is wide enough to accommodate anionic ssDNA, dsDNA or both. Biochemical analyses demonstrate that these five positively charged residues are essential for DNA binding and for RadA-catalyzed D-loop formation. We suggest that the overwound right-handed RadA filament represents a functional conformation in the homology search and pairing reaction. A new structural model is proposed for the homologous interactions between a RadA-ssDNA nucleoprotein filament and its dsDNA target.
Received: July 20, 2007; Accepted: August 16, 2007; Published: September 12, 2007
Figure 1. Crystal packing and quaternary structures.
(A) SsoRadA protomers packed into three extended helical filaments. Chain A was located at the origin of the unit cell, whereas chains B and C were located one-third and two-third diagonal to the unit cell. (B) Side view of the SsoRadA right-handed helical filament crystal structure. The helical pitch of the filament is 98 Å. Each protomer is shown in a different color. The N-terminal domain (NTD), polymerization motif (PM), and central ATPase domain are indicated. (C) The Phe73 of the PM is buried in the hydrophobic pocket of the neighboring ATPase domain. Several hydrophobic residues that interact with the Phe73 side chain are indicated. The interactions result in the assembly of SsoRadA protomers into a filament. 2Fo–Fc electron density maps (contoured at 1.0 σ), corresponding to the PM are shown in orange.
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