E-coli Rep oligomers are required to initiate DNA unwinding in vitro
E-coli Rep oligomers are required to initiate DNA unwinding in vitro
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DOI：
10.1006/jmbi.2001.4758
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发表时间：
2001-07-06
影响因子：
5.6
通讯作者：
Lohman, TM
中科院分区：
文献类型：
--
作者：
Cheng, W;Hsieh, J;Lohman, TM
Coli Rep蛋白是一个3‘到5’SF1超家族DNA解旋酶，在没有DNA的情况下是单体，但在结合单链或双链DNA时可以二聚化。各种生化研究表明，Rep二聚化对其解旋酶活性很重要；然而，最近对嗜热脂肪芽孢杆菌PcrA的结构研究导致了SF1解旋酶的功能，如E.coliRep和E.coliUvrD，其功能是单体解旋酶。通过对Rep催化的DNA解链的稳态前停流和化学猝灭动力学研究，我们研究了Rep齐聚对其DNA解旋酶活性是否重要的问题。四个独立实验的结果表明，在体外，DNA解旋酶活性的启动需要Rep寡聚。当只有Rep单体与DNA底物结合时，即使使用了荧光DNA底物，可以检测到ss-ds-DNA连接处前几个碱基对的部分解离，也没有观察到DNA解离。事实上，在这些条件下，ATP水解会导致Rep单体从DNA上解离，而不是DNA解离。这些研究表明，野生型Rep单体不能在体外启动DNA解离，需要寡聚。(C)2001年学术出版社。
coli Rep protein is a 3 ' to 5 ' SF1 superfamily DNA helicase which is monomeric in the absence of DNA, but can dimerize upon binding either single-stranded or duplex DNA. A variety of biochemical studies have led to proposals that Rep dimerization is important for its helicase activity; however, recent structural studies of Bacillus stearothermophilus PcrA have led to suggestions that SF1 helicases, such as E. coli Rep and E. coli UvrD, function as monomeric helicases. We have examined the question of whether Rep oligomerization is important for its DNA helicase activity using pre-steady state stopped-flow and chemical quenched-flow kinetic studies of Rep-catalyzed DNA unwinding. The results from four independent experiments demonstrate that Rep oligomerization is required for initiation of DNA helicase activity in vitro. No DNA unwinding is observed when only a Rep monomer is bound to the DNA substrate, even when fluorescent DNA substrates are used that can detect partial unwinding of the first few base-pairs at the ss-ds-DNA junction. In fact, under these conditions, ATP hydrolysis causes dissociation of the Rep monomer from the DNA, rather than DNA unwinding. These studies demonstrate that wild-type Rep monomers are unable to initiate DNA unwinding in vitro, and that oligomerization is required. (C) 2001 Academic Press.