Repair Mechanisms For Oxidative DNA Damage

为了生存，需氧生物代谢氧气以产生能量。在这个过程中，细胞产生活性氧（ROS）。ROS与所有细胞成分反应，包括脂质，蛋白质和DNA。这种氧化损伤与衰老过程和人类疾病，即癌症和神经变性有关。我们致力于确定修复氧化DNA损伤的生物化学和细胞过程。特别是，我们已经描绘了结构功能机制和生物学贡献的特定蛋白质，参与碱基切除修复（BER）途径。这一过程涉及DNA损伤的识别和切除，以及天然遗传物质的恢复。DNA修复缺陷会导致突变或细胞死亡，导致疾病的发展。 我们的大部分工作都涉及定义Ape 1的生化功能，Ape 1是修复DNA中脱碱基（AP）位点的主要人类蛋白质，这是一种常见的遗传损伤。我们已经证明，Ape 1有助于修复3？- DNA中的修饰，包括错配、磷酸基团、磷酸糖基和酪氨酰残基。我们最近的工作发现，Ape 1在复杂的、生物相关的DNA结构的单链区域的AP位点处切割，例如气泡和叉中间体。这些发现扩展了已知的由这种酶处理的底物库，并表明Ape 1可能与转录和/或复制相关的新功能。我们最近的工作集中在调节Ape 1修复活动的潜在机制。例如，我们已经证明了单链DNA结合蛋白RPA抑制Ape 1的混杂AP位点切割。此外，CSB，一种在人类早衰症Cockayne综合征中有缺陷的转录相关修复蛋白，被发现在转录泡中间体的AP位点激活Ape 1切割。最后，我们的研究发现，环境中的金属，铅，是一种有效的抑制剂Ape 1的活动，这表明这种重金属可能会引起其共同致癌作用的手段。我们目前正在设计方法来战略性地调节细胞中的Ape 1修复活性，希望开发更有效的抗癌治疗模式。 除了上述研究外，我们还启动了研究，以确定XRCC 1（一种主要的单链断裂修复（SSBR）因子）的生化和细胞贡献。这种蛋白质主要作为支架成分发挥作用，协调有效DNA修复所需的特定蛋白质-蛋白质相互作用。最近的工作已经确定了XRCC 1与人类神经退行性疾病AOA 1（Aprataxin）和SCAN 1（TDP 1）中有缺陷的蛋白质的关联。我们的研究表明XRCC 1通过与PCNA的相互作用与复制相联系，反对XRCC 1在BER早期步骤中的作用，并表明其与DNA聚合酶β的相互作用以及SSBR的后续步骤，特别是DNA切口连接的生物学相关作用。正在进行的使用动物模型（和衍生细胞）的研究正在确定XRCC 1和氧化DNA损伤修复与衰老和年龄相关疾病（即神经退行性疾病）的关系。此外，我们正在确定人类XRCC 1和相关蛋白变体对与疾病表现相关的受损细胞反应的贡献。

To live, aerobic organisms metabolize oxygen to generate energy. During this process, cells create reactive oxygen species (ROS). ROS react with all cellular constituents, including lipids, proteins, and DNA. Such oxidative damage has been associated with the aging process and human disease, namely cancer and neurodegeneration. We have worked to define the biochemical and cellular processes for repairing oxidative DNA damage. In particular, we have delineated the structure-function mechanisms and biological contributions of specific proteins that participate in the base excision repair (BER) pathway. This process involves the recognition and excision of DNA damage, and restoration of the native genetic material. Defects in DNA repair give rise to mutations or cell death, leading to the development of disease. Much of our effort has involved defining the biochemical functions of Ape1, the major human protein for repairing abasic (AP) sites in DNA, a frequent genetic damage. We have demonstrated that Ape1 contributes to the repair of 3?-modifications in DNA as well, including mismatches, phosphate groups, phosphogycolates, and tyrosyl residues. Our more recent work has found that Ape1 cleaves at AP sites in single-stranded regions of complex, biologically-relevant DNA structures, such as bubble and fork intermediates. These findings expand the known repertoire of substrates processed by this enzyme, and suggest novel functions for Ape1 likely coupled to transcription and/or replication. Our recent work has focused on potential mechanisms of regulating Ape1 repair activities. For instance, we have demonstrated that the single-stranded DNA binding protein RPA inhibits promiscuous AP site incision by Ape1. In addition, CSB, a transcription-related repair protein defective in the human premature aging disorder Cockayne Syndrome, was found to activate Ape1 cleavage at AP sites in transcription bubble intermediates. Finally, our studies have discovered that the environmental metal, lead, is a potent inhibitor of Ape1 activity, suggesting a means by which this heavy metal may elicit its co-carcinogenic effects. We are currently designing methods to strategically regulate Ape1 repair activity in cells in the hopes of developing more effective anti-cancer treatment paradigms. In addition to the investigations above, we have initiated studies to determine the biochemical and cellular contributions of XRCC1, a major single-strand break repair (SSBR) factor. This protein functions primarily as a scaffold component, orchestrating specific protein-protein interactions required for efficient DNA repair. Recent work has identified associations of XRCC1 with proteins defective in human neurodegenerative disorders AOA1 (Aprataxin) and SCAN1 (TDP1). Our studies suggest a link of XRCC1 to replication via an interaction with PCNA, argue against a role for XRCC1 in the early steps of BER, and indicate a biologically-relevant role for its interaction with DNA polymerase beta and in the subsequent steps of SSBR, specifically DNA nick ligation. Ongoing studies using animal models (and derived cells) are determining the relationship of XRCC1 and oxidative DNA damage repair to aging and age-related disease, namely neurodegeneration. Additionally, we are determining the contribution, if any, of human XRCC1 and associated protein variants to impaired cellular responses that are related to disease manifestation.