The role of DNA polymerase eta in DNA damage response and p53 activation

描述(申请人提供)：DNA聚合酶ETA在DNA损伤反应和P53激活中的作用。本研究旨在探讨DNA聚合酶ETA(PolH)在DNA损伤反应和P53激活中的信号通路，以及P53和PolH之间的信号通路相互作用对细胞存活和死亡的影响。为了描述P53在DNA损伤反应中的作用，我们发现DNA损伤可以以依赖于P53的方式诱导PolH。PolH是着色性干皮病(XP)基因的产物。XP是一种常染色体隐性遗传病，XP患者容易早发恶性皮肤癌。有趣的是，我们发现，PolH的敲除通过抑制DNA损伤诱导的细胞凋亡来提高细胞存活率。我们还发现，DNA损伤诱导的P53的激活在PolH基因敲除细胞和PolH零细胞中都受到损害，这可以被重组的PolH拯救。此外，我们还发现PolH通过ATM-Chk2-P53途径调节DNA损伤反应。最后，我们最近的初步研究表明，当DNA损伤时，PolH蛋白的稳定性降低，PolH与MDM2和Pirh2物理上相互作用，这两个基因都是P53靶基因和E3连接酶。综上所述，我们假设PolH活性受多条途径调节，PolH在DNA损伤反应和P53激活中具有新的功能。为了进一步解决这一问题，提出了以下三个具体目标：(1)确定PolH在基础和DNA损伤条件下是否以及如何调节PolH的表达；(2)确定PolH与MDM2或Pirh2相互作用的功能意义；(3)确定PolH在DNA损伤反应和P53激活中的作用。 公共卫生相关性：众所周知，P53抑癌基因的缺失会导致基因组不稳定，而着色性干皮病(XP)患者通常表现出很高的基因组不稳定频率。此外，在缺乏着色性干皮病(XP)和p53基因的细胞中，基因组不稳定的程度比单独缺乏其中任何一种基因的细胞要高得多。有趣的是，我们发现XPV基因产物PolH是一个新的P53靶基因，并且PolH的敲除通过抑制DNA损伤诱导的细胞凋亡来提高细胞存活率。令人惊讶的是，我们发现DNA损伤后P53的激活在PolH敲除细胞和PolH零细胞中都受到损害，这可以通过重组PolH来拯救。鉴于PolH的缺失使XP患者容易早发多种恶性皮肤癌，而且P53是一种肿瘤抑制因子，进一步研究PolH如何调节P53的激活和DNA损伤反应是非常有意义和必要的。

DESCRIPTION (provided by applicant): The role of DNA polymerase eta in DNA damage response and p53 activation. This application is proposed to address the signaling pathway of DNA polymerase eta (PolH) in DNA damage response and p53 activation and the effect of the signal pathway interaction between p53 and PolH on cell survival and death. In an effort to characterize the role of p53 in DNA damage response, we found that PolH can be induced by DNA damage in a p53-dependent manner. PolH is the product of the Xeroderma Pigmentosum (XP) gene. XP is an autosomal recessive disorder, and XP patients are prone to early onset of malignant skin cancers. Interestingly, we found that knockdown of PolH enhances cell survival by inhibiting DNA damage-induced apoptosis. We also found that DNA damage-induced activation of p53 is impaired in both PolH-knockdown and PolH-null cells, which can be rescued by a reconstituted PolH. Furthermore, we found that PolH modulates DNA damage response via the ATM- ChK2-p53 pathway. Finally, our recent preliminary studies showed that the stability of PolH protein is decreased upon DNA damage and PolH physically interacts with Mdm2 and Pirh2, both of which are a p53 target gene and an E3 ligase. Taken together, we hypothesize that PolH activity is regulated by multiple pathways and PolH has novel functions in DNA damage response and p53 activation. To further address this, the following three specific aims are proposed: (1) to determine whether and how PolH expression is regulated at basal and DNA damage conditions; (2) to determine the functional significance of the interaction between PolH and Mdm2 or Pirh2; and (3) to determine the role of PolH in DNA damage response and p53 activation. PUBLIC HEALTH RELEVANCE: It is well-known that loss of p53 tumor suppressor leads to genome instability and Xeroderma Pigmentosum (XP) patients often exhibit a high frequency of genome instability. In addition, the magnitude of genome instability is much higher in cells lacking both Xeroderma Pigmentosum (XP) and p53 genes than cells lacking either one individually. Interestingly, we found that PolH, the XPV gene product, is a novel p53 target gene and knockdown of PolH enhances cell survival by inhibiting DNA damage-induced apoptosis. Surprisingly, we found that activation of p53 following DNA damage is impaired in both PolH-knockdown and PolH-null cells, which can be rescued by reconstituted PolH. Given the fact that loss of PolH predisposes XP patients to early onset of multiple malignant skin cancers and that p53 is a tumor suppressor, further studies to address how PolH modulates p53 activation and DNA damage response are highly significant and warranted.