APE1 Cleavage Mechanisms during DNA Repair

暴露于环境危害诱导氧化应激并促进对细胞的有害修饰。 DNA的结构。这些修饰具有潜在的致突变性，并可促进许多人类疾病， 包括癌症碱基切除修复（BER）途径是细胞对DNA氧化的主要防御途径 破坏并维持基因组稳定性。在这一点上，关键BER酶的遗传多态性和缺陷 在几个人群中出现，并且通常与癌症风险增加有关。一个基本 BER酶是人脱嘌呤/脱嘧啶（AP）内切核酸酶1（APE 1），是一种多功能酶 在BER过程中处理DNA损伤利用相同的活性位点，APE 1执行AP核酸内切酶 核酸内切酶（endo）和3'至5'核酸外切酶（exo）活性。APE 1内切活性已被严格表征。在 相比之下，APE 1外切活性的机制仍然不清楚，也不清楚紧凑的活性位点如何能够 在一个实施方案中，所述第二碱基序列容纳内底物（无碱基位点）和外底物（3'错配或受损碱基）。 此外，在APE 1 exo活性期间，BER共复合物对毒性DNA中间体的通道作用仍然存在 完全未经研究，在我们对BER的理解中留下了很大的空白。因此，本提案的目的 目的是确定APE 1在修复错配和受损DNA末端过程中的外切机制。我们将会下 这种活性在BER底物通道作用过程中与较大的DNA修复共复合物有关。我们假设 APE 1的exo反应依赖于独特的活性位点接触， 校正和处理受损的DNA末端。我们还预测外切底物促进DNA BER期间APE 1和DNA聚合酶β（途径中的下一个酶）之间的底物通道。 为了验证这一点，我们提出了以下目标：（1）确定BER过程中APE 1 exo活性的机制 校正;（2）确定APE 1催化去除3′-PG末端损伤的机制;（3）确定 APE 1 exo活性过程中BER底物沟道的机制。为了实现这些目标，我们将利用 时间推移X射线晶体学观察在原子水平上的催化作用，以及前稳态酶动力学 解析出催化过程中重要步骤的速率。为了解决基板沟道的机制 在APE 1 exo活动期间，我们将使用单分子全内反射显微镜（TIRFM）观察 BER复合物在DNA上的组装/分解。小角中子散射将补充TIRFM 通过确定BER协复合物的结构包络进行研究。通过这种多学科的方法，我们 这将有助于揭示先前未充分研究的APE 1 DNA修复机制。有了这些信息，我们将 更接近我们的长期目标，即为合理的药物设计提供基础，以开发更多 靶向参与DNA损伤的蛋白质的有效化疗剂和协同药物组合 反应这种方法已被证明对DNA修复途径的核心蛋白质（如PARP-1）是成功的。

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.