Regulation of polymerase switching in translesion synthesis

描述（由申请人提供）：环境因素和内源性代谢过程产生聚合酶阻断DNA损伤，这可能会使DNA复制机制停止并导致基因组不稳定。为了避免这样的事件，细胞已经进化出一种称为translesion synthesis（TLS）的机制，该机制使用低保真度聚合酶来允许DNA合成穿过病变而无需实际修复。我们的长期目标是了解TLS的调控机制，并确定其在预防基因组不稳定性和肿瘤形成中的重要性。最近，我们的团队和其他人将Spartan（也称为DVC 1）确定为TLS的新型调节器。我们证明了Spartan耗尽导致易错TLS聚合酶Pol zeta的积累和更高的UV诱导的诱变。基于这些观察结果，我们现在假设Spartan负调节Pol zeta复合物，从而防止基因组不稳定和肿瘤形成。为了进一步了解Spartan对TLS的调控机制及其生理意义，我们拟：（1）研究Spartan对Polzeta的调控机制;（2）研究Spartan基因敲除细胞DNA损伤和细胞周期缺陷的原因;（3）研究Spartan在小鼠基因组稳定性和肿瘤抑制中的作用。目的1将集中在Spartan的一个假定的金属蛋白酶结构域SprT结构域，并通过表征其体外活性来确定其在Pol zeta调节中的作用。此外，我们将研究Pol zeta的调节剂Rev 1如何参与Pol zeta的复杂形成。在目标2中，我们将研究Spartan基因敲除细胞中DNA损伤和细胞周期缺陷的原因，并确定其是否与Spartan在TLS中的功能有关。最后，在目标3中，我们将使用小鼠模型确定Spartan是否对预防突变、基因组不稳定性和肿瘤形成很重要。总之，这些研究不仅将为Spartan对TLS的调控提供新的线索，还将为TLS调控在基因组维持和肿瘤抑制中的作用提供新的见解。

DESCRIPTION (provided by applicant): Environmental factors and endogenous metabolic processes create polymerase-blocking DNA lesions, which can stall DNA replication machinery and cause genome instability. To avoid such events, cells have evolved a mechanism called translesion synthesis (TLS), which uses low fidelity polymerases to allow DNA synthesis across the lesions without actual repair. Our long-term goal is to understand regulatory mechanisms of TLS and determine its importance in prevention of genome instability and tumor formation. Recently, our group and others identified Spartan (also known as DVC1) as a novel regulator of TLS. We demonstrated that Spartan depletion results in accumulation of the error-prone TLS polymerase Pol zeta and higher UV-induced mutagenesis. Building on these observations, we now hypothesize that Spartan negatively regulates Pol zeta complex, and thereby prevents genome instability and tumor formation. To gain more insights into the mechanism of TLS regulation by Spartan and its physiological relevance, we propose to: (1) Study the mechanism of Pol zeta regulation by Spartan, (2) Investigate the cause of DNA damage and cell-cycle defects in Spartan knockout cells, (3) Examine the role of Spartan in genome stability and tumor suppression in mouse. Aim 1 will focus on the SprT domain, a putative metalloprotease domain of Spartan, and determine its role in Pol zeta regulation by characterizing its activity in vitro. n addition, we will investigate how Rev1, a regulator of Pol zeta, is involved in the complex formation of Pol zeta. In Aim 2, we will investigate the cause of DNA damage and cell cycle defects in Spartan knockout cells and determine whether it is related to Spartan's function in TLS. Finally in Aim 3, we will determine whether Spartan is important to prevent mutations, genome instability and tumor formation using mouse models. In summary, these studies will not only shed new light on the regulation of TLS by Spartan, but also provide novel insights into the role of TLS regulation in genome maintenance and tumor suppression.