APE1 Cleavage Mechanisms during DNA Repair

暴露在环境危害中会诱导氧化应激，并促进对 DNA的结构。这些修饰具有潜在的突变性，可能会引发许多人类疾病， 包括癌症。碱基切除修复(BER)途径是细胞对氧化DNA的主要防御 破坏并维持基因组的稳定性。至此，关键的BER酶的遗传多态和缺陷 出现在几个人类群体中，并经常与癌症风险增加有关。必需品 BER酶是一种多功能酶，是人无嘌呤/脱嘧啶(AP)核酸内切酶1(APE1)。 在误码率过程中处理DNA损伤。利用相同的活性位点，APE1执行两种AP内切酶 (Endo)和3‘至5’核酸外切酶(Exo)活性。APE1Endo活性已经得到了严格的表征。在……里面 相比之下，APE1exo活性的机制仍然难以捉摸，也不清楚致密的活性部位如何 既可容纳内切底物(基本部位)，也可容纳外切底物(3‘不匹配或受损的碱基)。 此外，在ape1 exo活性期间，ber共复合体对有毒dna中间体的输送仍然存在。 完全没有研究，在我们对误码率的理解上留下了很大的空白。因此，这项提案的目标是 目的是确定APE1exo在错配和受损DNA末端修复过程中的作用机制。我们会把 这种活动是在BER底物通道期间较大的DNA修复共复合体的背景下进行的。我们假设 APE1的外周反应依赖于独特的活性部位接触来打开结合口袋 对受损DNA末端进行校对和处理。我们还预测外源底物能促进DNA 在BER过程中，APE1和DNA聚合酶β(该途径中的下一个酶)之间的底物通道。 为了验证这一点，我们提出了以下目标：(1)确定误码率过程中APE1exo活性的机制 校对；(2)确定APE1催化去除3‘-PG末端损伤的机理；(3)确定 APE1exo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 延时X射线结晶学在原子水平上观察催化作用，以及预稳态酶动力学 分析出催化过程中重要步骤的速率。为了解决衬底沟道的机制 在APE1exo活性过程中，我们将使用单分子全内反射显微镜(TIRFM)来观察 DNA上BER复合体的组装/拆卸。小角中子散射将是对TIRFM的补充 通过确定BER共复合体的结构包络进行研究。使用这种多学科的方法，我们 将揭示以前未被研究的APE1DNA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，以开发更多的药物 有效的化疗药物和针对参与DNA损伤的蛋白质的协同药物组合 回应。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.