Sleeth Kate M, Sørensen Claus Storgaard, Issaeva Natalia, Dziegielewski Jaroslaw, Bartek Jiri, Helleday Thomas
The Institute for Cancer Studies, University of Sheffield, Sheffield, UK.
J Mol Biol. 2007 Oct 12;373(1):38-47. doi: 10.1016/j.jmb.2007.07.068. Epub 2007 Aug 15.
The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role of RPA in homologous recombination in assembly of the RAD51 and RAD52 proteins. Furthermore, our data suggest that replacement of RPA with the RAD51 and RAD52 proteins is affected by checkpoint signalling.
复制蛋白A(RPA)参与了大多数(即便不是全部)涉及单链DNA的核代谢过程。在此,我们表明RPA在人类中通过同源重组修复系统参与停滞复制叉处的基因组维持。RPA蛋白的缺失抑制了羟基脲诱导的复制停滞后RAD51核灶的形成,导致持续存在未修复的DNA双链断裂(DSB)。我们证明了RPA在同源定向重组修复中具有直接作用。我们发现RPA对于检查点激酶1(Chk1)的激活是可有可无的,并且在复制应激时RPA直接结合RAD52,这表明其在重组修复中具有直接作用。此外,我们表明用UCN - 01抑制Chk1会减少RPA从染色质上的解离,并抑制RAD51和RAD52与DNA的结合。总之,我们的数据表明RPA在RAD51和RAD52蛋白组装的同源重组中具有直接作用。此外,我们的数据表明用RAD51和RAD52蛋白替代RPA受检查点信号传导影响。