Parplys Ann C, Kratz Katja, Speed Michael C, Leung Stanley G, Schild David, Wiese Claudia
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.
DNA Repair (Amst). 2014 Dec;24:87-97. doi: 10.1016/j.dnarep.2014.09.007. Epub 2014 Oct 5.
RAD51-associated protein 1 (RAD51AP1) is critical for homologous recombination (HR) by interacting with and stimulating the activities of the RAD51 and DMC1 recombinases. In human somatic cells, knockdown of RAD51AP1 results in increased sensitivity to DNA damaging agents and to impaired HR, but the formation of DNA damage-induced RAD51 foci is unaffected. Here, we generated a genetic model system, based on chicken DT40 cells, to assess the phenotype of fully inactivated RAD51AP1 in vertebrate cells. Targeted inactivation of both RAD51AP1 alleles has no effect on either viability or doubling-time in undamaged cells, but leads to increased levels of cytotoxicity after exposure to cisplatin or to ionizing radiation. Interestingly, ectopic expression of GgRAD51AP1, but not of HsRAD51AP1 is able to fully complement in cell survival assays. Notably, in RAD51AP1-deficient DT40 cells the resolution of DNA damage-induced RAD51 foci is greatly slowed down, while their formation is not impaired. We also identify, for the first time, an important role for RAD51AP1 in counteracting both spontaneous and DNA damage-induced replication stress. In human and in chicken cells, RAD51AP1 is required to maintain wild type speed of replication fork progression, and both RAD51AP1-depleted human cells and RAD51AP1-deficient DT40 cells respond to replication stress by a slow-down of replication fork elongation rates. However, increased firing of replication origins occurs in RAD51AP1-/- DT40 cells, likely to ensure the timely duplication of the entire genome. Taken together, our results may explain why RAD51AP1 commonly is overexpressed in tumor cells and tissues, and we speculate that the disruption of RAD51AP1 function could be a promising approach in targeted tumor therapy.
RAD51相关蛋白1（RAD51AP1）通过与RAD51和DMC1重组酶相互作用并刺激其活性，对同源重组（HR）至关重要。在人类体细胞中，敲低RAD51AP1会导致对DNA损伤剂的敏感性增加以及HR受损，但DNA损伤诱导的RAD51焦点的形成不受影响。在这里，我们基于鸡DT40细胞构建了一个遗传模型系统，以评估脊椎动物细胞中完全失活的RAD51AP1的表型。两个RAD51AP1等位基因的靶向失活对未受损细胞的活力或倍增时间均无影响，但在暴露于顺铂或电离辐射后会导致细胞毒性水平升高。有趣的是，GgRAD51AP1的异位表达而非HsRAD51AP1的异位表达能够在细胞存活测定中完全互补。值得注意的是，在RAD51AP1缺陷的DT40细胞中，DNA损伤诱导的RAD51焦点的分辨率大大减慢，而其形成并未受损。我们还首次确定了RAD51AP1在对抗自发和DNA损伤诱导的复制应激方面的重要作用。在人类和鸡细胞中，维持复制叉进展的野生型速度需要RAD51AP1，并且RAD51AP1耗尽的人类细胞和RAD51AP1缺陷的DT40细胞都通过复制叉延伸速率的减慢来应对复制应激。然而，RAD51AP1 - / - DT40细胞中复制起点的激发增加，这可能是为了确保整个基因组的及时复制。综上所述，我们的结果可能解释了为什么RAD51AP1在肿瘤细胞和组织中通常过度表达，并且我们推测破坏RAD51AP1功能可能是靶向肿瘤治疗中的一种有前景的方法。
