Mechanisms of damaged DNA replication in eukaryotes

描述(申请人提供)：突变在许多疾病的病因学中起着关键作用，如自闭症、精神分裂症、糖尿病和癌症。大多数突变发生在受损DNA的复制过程中，这一过程称为跨病变合成(TLS)。TLS是由专门的TLS聚合酶进行的，这种聚合酶已经进化成适应DNA损伤。关于TLS聚合酶的几个基本的、尚未回答的问题仍然存在。在目标1中，我们将解决以下问题：TLS聚合酶是如何招募到停滞的复制分叉上的？为了做到这一点，我们将使用系综和单分子结合分析以及稳态动力学。这些研究将揭示TLS聚合酶重新聚集到泛素修饰的增殖细胞核抗原(UbPCNA)的机制，泛素修饰的增殖细胞核抗原是停滞复制叉的关键组成部分。这些研究还将揭示这些相互作用所需的结构主题。在目标2中，我们将解决以下问题：如何选择TLS聚合酶以重新招募到特定的DNA损伤？为了做到这一点，我们将使用系综和单分子结合分析。这些研究将揭示TLS聚合酶选择的机制以及DNA损伤对这一过程的影响。在目标3中，我们将解决以下问题：TLS聚合酶是如何在停滞的复制叉处进行结构组织的？为了做到这一点，我们将使用X射线晶体学，计算 建模和仿真，以及各种距离测量的实验。这些研究将首次揭示TLS聚合酶在这些蛋白质-DNA复合体中的结构组织，并揭示不同的TLS聚合酶在TLS的多步骤过程中是如何协调的。

DESCRIPTION (provided by applicant): Mutations play a critical role in the etiology of many diseases, such as autism, schizophrenia, diabetes, and cancer. Most mutations arise during the replication of damaged DNA, a process called translesion synthesis (TLS). TLS is carried out by specialized TLS polymerases, which have evolved to accommodate DNA damage. Several fundamental, unanswered questions about TLS polymerases remain. In Aim 1, we will address the following question: how are TLS polymerases recruited to stalled replication forks? To do this, we will use both ensemble and single-molecule binding assays and steady state kinetics. These studies will reveal the mechanisms of TLS polymerase recruitment to ubiquitin-modified PCNA (UbPCNA), a key component of stalled replication forks. These studies will also reveal the structural motifs required for these interactions. In Aim 2, we will address the following question: how are TLS polymerases selected for recruitment to specific DNA lesions? To do this, we will use ensemble and single-molecule binding assay. These studies will reveal the mechanisms of TLS polymerase selection and the influence of the DNA lesion on this process. In Aim 3, we will address the following question: how are TLS polymerases structurally organized at stalled replication fork? To do this, we will use X-ray crystallography, computational modeling and simulations, and a variety of experimental distance measurements. These studies will provide the first glimpse of the structural organization of TLS polymerases within these protein-DNA complexes and will reveal how the different TLS polymerases are coordinated during the multi-step process of TLS.