APE1 Cleavage Mechanisms during DNA Repair

DNA 修复过程中 APE1 切割机制

基本信息

批准号：
10443576
负责人：
Bret D Freudenthal
金额：
$ 37.37万
依托单位：
UNIVERSITY OF KANSAS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-15 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10443576
关键词：
Active Sites Address Aphorisms Base Excision Repairs Binding Breast Catalysis Cells Colorectal Complement Complex DNA DNA Damage DNA Polymerase beta DNA Repair DNA Repair Enzymes DNA Repair Pathway DNA Structure DNA biosynthesis DNA lesion DNA-(apurinic or apyrimidinic site) lyase Defect Development Drug Combinations Drug Design EXO1 gene Environmental Exposure Environmental Hazards Enzyme Kinetics Enzymes Excision Exonuclease Exposure to Genetic Polymorphism Genome Genome Stability Genomic Instability Goals Hand Health Human Kinetics Lead Light Malignant Neoplasms Mediating Microscopy Mismatch Repair Modification Molecular Monitor Multiprotein Complexes Mutation Neutrons Ovarian Oxidative Stress Pathway interactions Phosphodiesterase I Play Polymerase Population Positioning Attribute Process Proteins Reaction Reactive Oxygen Species Repair Complex Role Site Source Structure System Techniques Testing Time Variant Vertebral column X-Ray Crystallography base cancer risk cancer therapy endonuclease experimental study human disease interdisciplinary approach novel novel strategies oxidative DNA damage phosphoglycolate rational design repair enzyme repaired response single molecule therapeutic target

项目摘要

Exposure to environmental hazards induces oxidative stress and promotes deleterious modifications to the structure of DNA. These modifications are potentially mutagenic and can promote numerous human maladies, including cancer. The base excision repair (BER) pathway is the cells primary defense against oxidative DNA damage and maintains genome stability. To this point, genetic polymorphisms and defects in key BER enzymes show up in several human populations, and are often associated with an increased cancer risk. An essential BER enzyme is human apurinic/apyrimidinic (AP) endonuclease 1 (APE1), which is a multifunctional enzyme that processes DNA damage during BER. Utilizing the same active site, APE1 performs both AP endonuclease (endo) and 3' to 5' exonuclease (exo) activities. APE1 endo activity has been rigorously characterized. In contrast, the mechanism for APE1 exo activity remains elusive, and it is unclear how the compact active site can accommodate both an endo substrate (abasic site) and an exo substrate (3' mismatched or damaged base). Moreover, the channeling of toxic DNA intermediates by the BER co-complex during APE1 exo activities remains entirely unstudied, leaving a significant gap in our understanding of BER. Therefore, the objective of this proposal is to determine the APE1 exo mechanism during repair of mismatched and damaged DNA ends. We will place this activity in context of the larger DNA repair co-complex during BER substrate channeling. We hypothesize the exo reaction of APE1 is dependent on unique active site contacts to open the binding pocket during proofreading and the processing of damaged DNA ends. We additionally predict exo substrates promote DNA substrate channeling between APE1 and DNA polymerase beta (the next enzyme in the pathway) during BER. To test this, we propose the following aims: (1) Determine the mechanism of APE1 exo activity during BER proofreading; (2) Determine the mechanism of APE1 catalyzed removal of 3′-PG end damage; and (3) Determine the mechanism of BER substrate channeling during APE1 exo activity. To accomplish these aims we will utilize time-lapse X-ray crystallography to observe catalysis at the atomic level, and pre-steady-state enzyme kinetics to parse out the rates of important steps during catalysis. To address the mechanism of substrate channeling during APE1 exo activity, we will use single-molecule total internal reflection microscopy (TIRFM) to observe the assembly/disassembly of BER complexes on DNA. Small angle neutron scattering will complement the TIRFM studies by determining a structural envelope of the BER co-complex. Using this multidisciplinary approach, we will cast light on previously understudied APE1 DNA repair mechanisms. With this information in hand, we will be closer to our long-term goal of providing a basis for rational drug design towards the development of more effective chemotherapeutics and synergistic drug combinations that target proteins involved in the DNA damage response. This approach has proven successful for proteins central to DNA repair pathways, such as PARP-1.

暴露于环境危害会引起氧化应激，并促进有害的修改 DNA的结构。这些修饰是潜在的诱变，可以促进许多人类疾病，包括癌症。基本惊喜修复（BER）途径是细胞对氧化DNA的主要防御损害并保持基因组稳定性。至此，关键BER酶中的遗传多态性和缺陷出现在几个人口中，通常与癌症风险增加有关。必不可少的 BER酶是人肾上腺素/丙酰胺（AP）核酸内切酶1（APE1），这是一种多功能酶这会处理BER期间的DNA损伤。利用相同的活动站点，APE1执行两种AP内核酸酶（endo）和3'至5'核酸酶（EXO）活动。 APE1 ENDO活动已被严格表征。在对比，APE1 EXO活性的机制仍然难以捉摸，尚不清楚紧凑的活性位点如何容纳内托基材（脓肿部位）和EXO底物（3'不匹配或损坏的碱基）。此外，在APE1 EXO活动期间，BER共络合物对有毒DNA中间体的传递仍然存在完全没有研究，在我们对BER的理解上留下了很大的差距。因此，该提议的目的是为了在修复不匹配和受损的DNA末端修复过程中确定APE1 EXO机制。我们将放置在BER底物通道期间，在较大的DNA修复综合体的背景下，这种活性。我们假设 APE1的EXO反应取决于独特的主动位点触点，以打开结合口袋校对和处理受损的DNA末端的处理。我们还预测EXO底物促进DNA 在BER期间，APE1和DNA聚合酶β（途径中的下一个酶）之间的底物在BER之间传递。为了测试这一点，我们提出以下目的：（1）确定BER期间APE1 EXO活动的机制校对；（2）确定APE1的机理催化了3'-PG最终损伤的去除；（3）确定在APE1 EXO活性期间，BER底物引导的机理。为了实现这些目标，我们将利用延时X射线晶体学以观察原子水平的催化和稳态的酶动力学解析催化过程中重要步骤的速率。解决底物通道的机制在APE1 EXO活性期间，我们将使用单分子总内部反射显微镜（TIRFM）观察 DNA上Ber复合物的组装/拆卸。小角度中子散射将完成TIRFM 通过确定BER共复合的结构包膜来进行研究。使用这种多学科方法，我们将阐明先前理解的APE1 DNA修复机制。有了这些信息，我们将要更接近我们的长期目标，即提供理性药物设计的基础，以开发更多有效的化学治疗剂和协同药物组合，靶向参与DNA损伤的蛋白回复。事实证明，这种方法对于DNA修复途径（例如PARP-1）中心的蛋白质已被证明是成功的。