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中起关键作用，例如自闭症，精神分裂症，糖尿病和癌症。在损坏的DNA复制过程中，大多数突变都会出现，这一过程称为translesion合成（TLS）。 TLS由专门的TLS聚合酶进行，该聚合酶已进化以适应DNA损伤。关于TLS聚合酶的几个基本，未解决的问题仍然存在。在AIM 1中，我们将解决以下问题：如何将TLS聚合酶招募到停滞的复制叉？为此，我们将同时使用集合和单分子结合测定和稳态动力学。这些研究将揭示TLS聚合酶募集到泛素修饰的PCNA（UBPCNA）的机制，这是停滞的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AIM 2中，我们将解决以下问题：如何选择用于募集特定DNA病变的TLS聚合酶？为此，我们将使用集合和单分子结合测定法。这些研究将揭示TLS聚合酶选择的机制以及DNA病变对此过程的影响。在AIM 3中，我们将解决以下问题：在停滞的复制叉结构上如何组织TLS聚合酶？为此，我们将使用X射线晶体学，计算 建模和模拟，以及各种实验距离测量。这些研究将对这些蛋白质-DNA复合物中TLS聚合酶的结构组织提供第一眼，并将揭示在TLS多步骤过程中如何协调不同的TLS聚合酶。