Menetski J P, Varghese A, Kowalczykowski S C
Department of Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611.
Biochemistry. 1988 Feb 23;27(4):1205-12. doi: 10.1021/bi00404a021.
The properties of the high-affinity single-stranded DNA (ssDNA) binding state of Escherichia coli recA protein have been studied. We find that all of the nucleoside triphosphates that are hydrolyzed by recA protein induce a high-affinity ssDNA binding state. The effect of ATP binding to recA protein was partially separated from the ATP hydrolytic event by substituting calcium chloride for magnesium chloride in the binding buffer. Under these conditions, the rate of ATP hydrolysis is greatly inhibited. ATP increases the affinity of recA protein for ssDNA in a concentration-dependent manner in the presence of both calcium and magnesium chloride with apparent Kd values of 375 and 500 microM ATP, respectively. Under nonhydrolytic conditions, the molar ratio of ATP to ADP has an effect on the recA protein ssDNA binding affinity. Over an ATP/ADP molar ratio of 2-3, the affinity of recA protein for ssDNA shifts cooperatively from a low-to a high-affinity state.
对大肠杆菌recA蛋白高亲和力单链DNA（ssDNA）结合状态的特性进行了研究。我们发现，recA蛋白水解的所有核苷三磷酸都会诱导高亲和力ssDNA结合状态。通过在结合缓冲液中用氯化钙替代氯化镁，将ATP与recA蛋白结合的效应与ATP水解事件部分分离。在这些条件下，ATP水解速率受到极大抑制。在氯化钙和氯化镁同时存在的情况下，ATP以浓度依赖的方式增加recA蛋白对ssDNA的亲和力，ATP的表观解离常数（Kd）值分别为375和500微摩尔。在非水解条件下，ATP与ADP的摩尔比对recA蛋白与ssDNA的结合亲和力有影响。在ATP/ADP摩尔比超过2至3时，recA蛋白对ssDNA的亲和力从低亲和力状态协同转变为高亲和力状态。
