Roles of Spartan in translesion synthesis and UV-induced carcinogenesis

 描述（由申请方提供）：本研究旨在了解Spartan在跨病变合成（TLS）和UV诱导的致癌作用中的作用。TLS是一种复制后修复途径，在DNA复制过程中介导绕过阻碍复制叉的大体积DNA损伤。DNA病变的旁路由专门的低保真度TLS聚合酶介导，该聚合酶可以有效地复制扭曲或庞大的DNA加合物，留下待在稍后时间点修复的病变。TLS中的关键步骤是TLS聚合酶对复制性聚合酶的转换，这被认为是由依赖于RAD 18的PCNA泛素化（ubiquitination of PCNA，ub-PCNA）介导的。然而，确切的机制尚不清楚。我们将Spartan确定为TLS的关键监管者。Spartan的耗尽使细胞对UV损伤敏感，导致PCNA单泛素化减少，这伴随着RAD 18染色质缔合和RAD 18定位于DNA损伤位点的减少。有趣的是，Spartan在正常条件下与复制型DNA聚合酶结合，但在UV损伤时优先与TLS聚合酶POLH结合。根据我们的观察，我们提出Spartan是TLS的关键调节因子，需要在DNA损伤位点稳定RAD 18和ub-PCNA，并且可能直接调节TLS期间从复制聚合酶到TLS聚合酶的转换。然而，Spartan如何介导其调节功能以及TLS期间DNA聚合酶之间的切换尚不清楚，将是本研究的重点。本提案的具体目的是：目的1）确定Spartan在紫外线损伤后TLS中的功能;目的2）确定Spartan介导的TLS在紫外线诱导的致癌作用和对化疗药物的耐药性中的作用;目的3）确定Spartan的体内生理功能。 这项拟议的研究不仅将为TLS过程的调控机制提供有用的见解，而且还将探索靶向Spartan用于癌症治疗的潜力。

 DESCRIPTION (provided by applicant): This study aims at understanding the roles of Spartan in translesion synthesis (TLS) and UV-induced carcinogenesis. TLS is a post-replication repair pathway, which mediates bypass of bulky DNA lesions that stall replication forks, during DNA replication. Bypass of DNA lesions is mediated by specialized low-fidelity TLS polymerase that can replicate over distortions or bulky DNA adducts efficiently, leaving behind the lesion to be repaired at a later time point. The critical step in TLS is the switch of the replicative polymeras with TLS polymerase, which is proposed to be mediated by RAD18-dependent ubiquitination of PCNA (ub-PCNA). However, the exact mechanism is not clear. We identified Spartan as a key regulator of TLS. Depletion of Spartan renders cells sensitive to UV damage, results in a decrease in PCNA mono-ubiquitination, which is accompanied by a reduction of RAD18 chromatin association and RAD18 localization to DNA damage sites. Interestingly, Spartan binds to replicative DNA polymerase under normal conditions, but preferentially associates with TLS polymerase POLH upon UV damage. Based on our observations, we proposed that Spartan is a key regulator of TLS, required to stabilize RAD18 and ub-PCNA at the sites of DNA damage and may directly regulate the switch from replicative polymerase to TLS polymerase during TLS. However, how Spartan mediates its regulatory functions and the switch between the DNA polymerases during TLS is not known and will be the focus of this study. The specific aims for this proposal are: Aim 1) Determine the function of Spartan in TLS upon UV damage; Aim 2) Determine the roles of Spartan mediated TLS in UV-induced carcinogenesis and resistance to chemotherapeutic agents; Aim 3) Determine the in vivo physiological functions of Spartan. This proposed study will not only provide useful insights into the regulatory mechanism of TLS process, but also, will explore the potential of targeting Spartan for cancer therapy.