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会造成聚合酶阻断的DNA损伤，这可能会阻碍DNA复制机制，并导致基因组不稳定。为了避免此类事件，细胞进化出了一种名为跨病变合成(TLS)的机制，它使用低保真聚合酶来允许在没有实际修复的情况下跨病变合成DNA。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确定Spartan(也称为DVC1)是一种新的TLS调节因子。我们证明，斯巴达枯竭会导致容易出错的TLS聚合酶Pol Zeta的积累，并导致更高的紫外线诱变。在这些观察的基础上，我们现在假设斯巴达对Pol Zeta复合体进行负面调节，从而防止基因组不稳定和肿瘤形成。为了更深入地了解斯巴达对TLS的调节机制及其生理意义，我们建议：(1)研究斯巴达对Pol Zeta的调节机制；(2)研究斯巴达基因敲除细胞DNA损伤和细胞周期缺陷的原因；(3)检测斯巴达在小鼠基因组稳定和肿瘤抑制中的作用。目的1研究斯巴达金属蛋白酶结构域--SPRT结构域，并通过测定其体外活性确定其在Pol Zeta调控中的作用。此外，我们还将调查Pol Zeta的调节因子Rev1如何参与Pol Zeta的复杂形成。在目标2中，我们将研究斯巴达基因敲除细胞DNA损伤和细胞周期缺陷的原因，并确定这是否与斯巴达在TLS中的作用有关。最后，在目标3中，我们将通过小鼠模型确定斯巴达对于防止突变、基因组不稳定和肿瘤形成是否重要。综上所述，这些研究不仅将为斯巴达对TLS的调控提供新的线索，也将为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.