Cheng W, Hsieh J, Brendza K M, Lohman T M
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110, USA.
J Mol Biol. 2001 Jul 6;310(2):327-50. doi: 10.1006/jmbi.2001.4758.
E. 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.
大肠杆菌Rep蛋白是一种3'至5'的SF1超家族DNA解旋酶，在没有DNA的情况下呈单体状态，但在结合单链或双链DNA时可二聚化。各种生化研究表明，Rep二聚化对其解旋酶活性很重要；然而，最近嗜热脂肪芽孢杆菌PcrA的结构研究表明，SF1解旋酶，如大肠杆菌Rep和大肠杆菌UvrD，作为单体解旋酶发挥作用。我们使用Rep催化的DNA解旋的预稳态停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，Rep寡聚化是体外启动DNA解旋酶活性所必需的。当只有一个Rep单体与DNA底物结合时，未观察到DNA解旋，即使使用荧光DNA底物，该底物可以检测ss-ds-DNA连接处最初几个碱基对的部分解旋。事实上，在这些条件下，ATP水解导致Rep单体从DNA上解离，而不是DNA解旋。这些研究表明，野生型Rep单体在体外无法启动DNA解旋，并且需要寡聚化。
