Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
J Biol Chem. 2010 Jan 29;285(5):3211-26. doi: 10.1074/jbc.M109.028951. Epub 2009 Nov 12.
Disassembly of RecA protein subunits from a RecA filament has long been known to occur during DNA strand exchange, although its importance to this process has been controversial. An Escherichia coli RecA E38K/DeltaC17 double mutant protein displays a unique and pH-dependent mutational separation of DNA pairing and extended DNA strand exchange. Single strand DNA-dependent ATP hydrolysis is catalyzed by this mutant protein nearly normally from pH 6 to 8.5. It will also form filaments on DNA and promote DNA pairing. However, below pH 7.3, ATP hydrolysis is completely uncoupled from extended DNA strand exchange. The products of extended DNA strand exchange do not form. At the lower pH values, disassembly of RecA E38K/DeltaC17 filaments is strongly suppressed, even when homologous DNAs are paired and available for extended DNA strand exchange. Disassembly of RecA E38K/DeltaC17 filaments improves at pH 8.5, whereas complete DNA strand exchange is also restored. Under these sets of conditions, a tight correlation between filament disassembly and completion of DNA strand exchange is observed. This correlation provides evidence that RecA filament disassembly plays a major role in, and may be required for, DNA strand exchange. A requirement for RecA filament disassembly in DNA strand exchange has a variety of ramifications for the current models linking ATP hydrolysis to DNA strand exchange.
RecA 蛋白亚基从 RecA 丝的解组装在 DNA 链交换过程中早已为人所知，尽管其对该过程的重要性一直存在争议。一种大肠杆菌 RecA E38K/DeltaC17 双突变蛋白表现出独特的和 pH 依赖性的 DNA 配对和延伸 DNA 链交换的突变分离。从 pH 6 到 8.5，这种突变蛋白几乎正常地催化单链 DNA 依赖性 ATP 水解。它也会在 DNA 上形成丝并促进 DNA 配对。然而，在 pH 低于 7.3 时，ATP 水解与延伸的 DNA 链交换完全解偶联。不会形成延伸的 DNA 链交换产物。在较低的 pH 值下，RecA E38K/DeltaC17 丝的解组装受到强烈抑制，即使同源 DNA 配对并可用于延伸的 DNA 链交换。在 pH 8.5 时，RecA E38K/DeltaC17 丝的解组装得到改善，而完全的 DNA 链交换也得到恢复。在这些条件下，观察到丝解组装与 DNA 链交换完成之间的紧密相关性。这种相关性提供了证据，表明 RecA 丝解组装在 DNA 链交换中起着主要作用，并且可能是 DNA 链交换所必需的。DNA 链交换中 RecA 丝解组装的要求对将 ATP 水解与 DNA 链交换联系起来的当前模型有多种影响。
