McIlwraith M J, Van Dyck E, Masson J Y, Stasiak A Z, Stasiak A, West S C
Clare Hall Laboratories, Imperial Cancer Research Fund, South Mimms, Hertfordshire, EN6 3LD, UK.
J Mol Biol. 2000 Nov 24;304(2):151-64. doi: 10.1006/jmbi.2000.4180.
The human Rad51 recombinase is essential for the repair of double-strand breaks in DNA that occur in somatic cells after exposure to ionising irradiation, or in germ line cells undergoing meiotic recombination. The initiation of double-strand break repair is thought to involve resection of the double-strand break to produce 3'-ended single-stranded (ss) tails that invade homologous duplex DNA. Here, we have used purified proteins to set up a defined in vitro system for the initial strand invasion step of double-strand break repair. We show that (i) hRad51 binds to the ssDNA of tailed duplex DNA molecules, and (ii) hRad51 catalyses the invasion of tailed duplex DNA into homologous covalently closed DNA. Invasion is stimulated by the single-strand DNA binding protein RPA, and by the hRad52 protein. Strikingly, hRad51 forms terminal nucleoprotein filaments on either 3' or 5'-ssDNA tails and promotes strand invasion without regard for the polarity of the tail. Taken together, these results show that hRad51 is recruited to regions of ssDNA occurring at resected double-strand breaks, and that hRad51 shows no intrinsic polarity preference at the strand invasion step that initiates double-strand break repair.
人类Rad51重组酶对于修复DNA双链断裂至关重要,这种断裂发生在体细胞受到电离辐射后,或发生在进行减数分裂重组的生殖细胞中。双链断裂修复的起始被认为涉及双链断裂的切除,以产生3'端单链(ss)尾巴,该尾巴侵入同源双链DNA。在这里,我们使用纯化的蛋白质建立了一个确定的体外系统,用于双链断裂修复的初始链侵入步骤。我们表明:(i)hRad51与带尾巴的双链DNA分子的ssDNA结合;(ii)hRad51催化带尾巴的双链DNA侵入同源共价闭合DNA。单链DNA结合蛋白RPA和hRad52蛋白可刺激侵入。令人惊讶的是,hRad51在3'或5'-ssDNA尾巴上形成末端核蛋白丝,并促进链侵入,而不考虑尾巴的极性。综上所述,这些结果表明hRad51被招募到切除的双链断裂处出现的ssDNA区域,并且hRad51在启动双链断裂修复的链侵入步骤中没有内在的极性偏好。