Kornberg A, Scott J F, Bertsch L L
J Biol Chem. 1978 May 10;253(9):3298-304.
Hydrolysis of ATP by rep protein proceeds in the presence of a single-stranded region of DNA 4 residues long, but the true effector for rep ATPase appears to be a replicating fork rather than a random coil. At or near a fork in duplex DNA, rep ATPase action is different from what it is on DNA lacking secondary structure (single-stranded): (i) Km for ATP is lower, (ii) specificity is for ATP and dATP with no action on other nucleoside triphosphates, (iii) sensitivity to certain ATP analogs is reduced, (iv) presence of a DNA-nicking enzyme (e.g. cistron A protein induced by phiX174) is required, and (v) Escherichia coli DNA binding protein facilitates rather than inhibits. During the separation of strands accompanying replication, 2 molecules of nucleoside triphosphate (ATP or dATP) are hydrolyzed for every nucleotide polymerized. Utilization of ATP by rep protein may provide energy for catalytic strand separation at a fork in advance of replication.
Rep蛋白对ATP的水解作用在一段4个残基长的DNA单链区域存在时进行，但Rep ATP酶的真正效应物似乎是复制叉而非无规卷曲。在双链DNA的复制叉处或其附近，Rep ATP酶的作用与在缺乏二级结构的DNA（单链）上的作用不同：（i）ATP的米氏常数较低；（ii）对ATP和dATP具有特异性，对其他核苷三磷酸无作用；（iii）对某些ATP类似物的敏感性降低；（iv）需要一种DNA切口酶（如由phiX174诱导的顺反子A蛋白）；（v）大肠杆菌DNA结合蛋白起促进而非抑制作用。在伴随复制的链分离过程中，每聚合一个核苷酸，就有2分子核苷三磷酸（ATP或dATP）被水解。Rep蛋白对ATP的利用可能为复制前复制叉处的催化链分离提供能量。
