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首页> 外文期刊>Journal of Molecular Biology >E-coli Rep oligomers are required to initiate DNA unwinding in vitro
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E-coli Rep oligomers are required to initiate DNA unwinding in vitro

机译：需要E-coli Rep寡聚物才能启动体外DNA解链

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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. [References: 37]
机译：coli Rep蛋白是3'至5'SF1超家族DNA解旋酶，在不存在DNA的情况下为单体，但在结合单链或双链DNA时可以二聚。各种各样的生化研究提出了Rep二聚化对其解旋酶活性很重要的提议。然而，最近对嗜热脂肪芽孢杆菌PcrA的结构研究已经提示SF1解旋酶，如大肠杆菌Rep和大肠杆菌UvrD，起单体解旋酶的作用。我们已经通过Rep催化的DNA展开的稳态前停止流和化学淬灭流动力学研究，研究了Rep寡聚化对其DNA解旋酶活性是否重要的​​问题。四个独立实验的结果表明，体外启动DNA解旋酶活性需要Rep寡聚。当仅将Rep单体与DNA底物结合时，即使使用荧光DNA底物可检测到ss-ds-DNA交界处的前几个碱基对的部分解旋，也未观察到DNA的解旋。实际上，在这些条件下，ATP水解会导致Rep单体从DNA上解离，而不是DNA解链。这些研究表明，野生型Rep单体无法在体外启动DNA解链，并且需要低聚。 （C）2001学术出版社。 [参考：37]

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