Mechanisms of damaged DNA replication in eukaryotes

真核生物 DNA 复制受损的机制

• 批准号: 9297313
• 负责人: M. TODD WASHINGTON
• 金额: $ 31.46万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2018-09-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9297313
• 关键词: Address; Autistic Disorder; Binding; Biological Assay; Cancer Etiology; Complex; Computer Simulation; Crystallization; DNA; DNA Damage; DNA Replication Damage; DNA lesion; DNA replication fork; DNA-Directed DNA Polymerase; Development; Diabetes Mellitus; Disease; Enzymes; Etiology; Eukaryota; Funding; Germ Cells; Kinetics; Laboratories; Lesion; Malignant Neoplasms; Measurement; Modeling; Mutation; Play; Polymerase; Process; Proliferating Cell Nuclear Antigen; Proteins; Recruitment Activity; Roentgen Rays; Role; Schizophrenia; Somatic Cell; Structure; Ubiquitin; X-Ray Crystallography; flexibility; innovation; novel strategies; public health relevance; role model; scaffold; single molecule

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.

描述（由申请人提供）：突变在许多疾病的病因学中起关键作用，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，这一过程称为跨损伤合成（TLS）。TLS是由专门的TLS聚合酶进行的，这些聚合酶已经进化到适应DNA损伤。关于TLS聚合酶的几个基本的，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聚合酶如何被招募到停滞的复制叉？要做到这一点，我们将使用合奏和单分子结合试验和稳态动力学。这些研究将揭示TLS聚合酶招募泛素修饰的PCNA（UbPCNA）的机制，这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基序。在目标2中，我们将解决以下问题：如何选择TLS聚合酶用于招募特定的DNA损伤？为此，我们将使用系综和单分子结合测定。这些研究将揭示TLS聚合酶选择的机制以及DNA损伤对该过程的影响。在目标3中，我们将解决以下问题：TLS聚合酶在停滞的复制叉中的结构是如何组织的？为此，我们将使用X射线晶体学，计算 建模和仿真，以及各种实验距离测量。这些研究将首次揭示这些蛋白质-DNA复合物中TLS聚合酶的结构组织，并揭示不同TLS聚合酶在TLS的多步骤过程中是如何协调的。