RAD51AP1-deficiency in vertebrate cells impairs DNA replication.
RAD51AP1-deficiency in vertebrate cells impairs DNA replication.
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DOI：
10.1016/j.dnarep.2014.09.007
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发表时间：
2014-12
期刊：
影响因子：
3.8
通讯作者：
Wiese, Claudia
中科院分区：
文献类型：
--
作者：
Parplys, Ann C.;Kratz, Katja;Speed, Michael C.;Leung, Stanley G.;Schild, David;Wiese, Claudia
RAD 51相关蛋白1（RAD 51 AP 1）通过与RAD 51和DMC 1重组酶相互作用并刺激其活性而对同源重组（HR）至关重要。在人体细胞中，敲低RAD 51 AP 1导致对DNA损伤剂和受损HR的敏感性增加，但DNA损伤诱导的RAD 51灶的形成不受影响。在这里，我们产生了一个遗传模型系统，基于鸡DT 40细胞，以评估在脊椎动物细胞中完全失活的RAD 51 AP 1的表型。两个RAD 51 AP 1等位基因的靶向失活对未受损细胞的活力或倍增时间没有影响，但暴露于顺铂或电离辐射后导致细胞毒性水平增加。有趣的是，在细胞存活测定中，GgRAD 51 AP 1的异位表达而不是HsRAD 51 AP 1的异位表达能够完全互补。值得注意的是，在RAD 51 AP 1缺陷的DT 40细胞中，DNA损伤诱导的RAD 51病灶的分辨率大大减慢，而它们的形成没有受损。我们还确定，第一次，一个重要的作用，RAD 51 AP 1在抵消自发和DNA损伤诱导的复制压力。在人类和鸡细胞中，需要RAD 51 AP 1来维持复制叉进展的野生型速度，并且RAD 51 AP 1耗尽的人类细胞和RAD 51 AP 1缺陷型DT 40细胞都通过减慢复制叉延伸速率来响应复制应激。然而，在RAD 51 AP 1 −/− DT 40细胞中，复制起点的发射增加，可能确保整个基因组的及时复制。综上所述，我们的研究结果可以解释为什么RAD 51 AP 1通常在肿瘤细胞和组织中过表达，我们推测RAD 51 AP 1功能的破坏可能是靶向肿瘤治疗的一种有前途的方法。
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.
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