Interplay of base excision repair and DNA damage response for genome maintenance

碱基切除修复和 DNA 损伤反应在基因组维护中的相互作用

• 批准号: 9248805
• 负责人: Annie J McPherson
• 金额: $ 4.4万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9248805
• 关键词: Address; Affinity Chromatography; Base Excision Repairs; Binding Proteins; Biological; Cancer Etiology; Cause of Death; Cell Cycle Arrest; Cell Cycle Regulation; Cell Nucleus; Cell Respiration; Cells; Chromatin; Colon Carcinoma; Communication; Complement; DNA; DNA Damage; DNA Maintenance; DNA Repair; DNA Repair Gene; DNA Repair Pathway; DNA damage checkpoint; DNA lesion; DNA-Binding Proteins; Data; Defect; Development; Disease; End Point Assay; Ensure; Event; Exhibits; Genetic; Genetic Materials; Genome; Goals; Human; Knowledge; Lead; Left; Lesion; Link; Lyase; Maintenance; Malignant - descriptor; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mitochondria; Modeling; Modification; Mutagenesis; Mutation; Normal Cell; Nuclear; Organelles; Orthologous Gene; Oxides; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Play; Post-Translational Protein Processing; Proteins; Reactive Oxygen Species; Recombinants; Recruitment Activity; Regulation; Reporting; Research Personnel; Role; Saccharomyces cerevisiae; Saccharomycetales; Signal Transduction; Site; Source; Testing; Time; Ubiquitin; United States; Variant; Work; Yeasts; activator 1 protein; arginyllysine; base; cell growth; cell injury; combat; experimental study; genome integrity; in vitro Assay; insight; malignant stomach neoplasm; man; mitochondrial genome; mutant; new therapeutic target; novel strategies; novel therapeutic intervention; oxidative DNA damage; oxidative damage; prevent; public health relevance; repaired; response; tumor progression

 DESCRIPTION (provided by applicant): Cancer is one of the leading causes of death in the United States. Genetic instability is an enabling cancer hallmark that facilitates the accumulation of progressively more malignant cancer phenotypes. To prevent genetic instability, cells have developed multiple evolutionarily conserved DNA damage repair and DNA damage response pathways that protect both the nuclear and mitochondrial genomes. One major cause of DNA damage is oxidative DNA damage, which results from reactive oxygen species (ROS). The major repair pathway for oxidative DNA damage is the Base Excision Repair (BER) pathway which is conserved from yeast to man. Although the BER pathway plays a major role in protecting both the nuclear and mitochondrial genomes, how this pathway is regulated is not known. Our recent studies have addressed this gap in knowledge by focusing on S. cerevisiae Ntg1, an evolutionarily conserved BER protein that recognizes and excises oxidized base lesions. The human Ntg1 counterpart, Nthl1, has altered function/localization that is linked to both colon and gastric cancers highlighting the importance of Nthl1 in preventing cancer and of defining mechanisms that regulate the BER pathway. Our work reveals that both Ntg1 and Nthl1 can be targeted to the nucleus or mitochondria in response to organelle-specific DNA damage. Consistent with a model where posttranslational modification(s) of BER proteins contribute to relocalization and/or regulation, our preliminary studies in budding yeast reveal that the nuclear pool of Ntg1 is modified by the Small Ubiquitin-like Modifier (SUMO) following nuclear oxidative damage. Importantly, preliminary data show that human Nthl1 can also be SUMO modified. To assess the functional importance of SUMO modification of these BER proteins, we have mapped all the SUMO modification sites on Ntg1 (K20,38,376,388,396) and created a non-sumoylatable (K->R) ntg1 variant (ntg1∆SUMO). Although recombinant ntg1∆SUMO retains catalytic activity in an in vitro assay, cells expressing ntg1∆SUMO as the sole copy of Ntg1 exhibit a defect in the DNA damage response failing to arrest the cell cycle in response to DNA damage. Based on this preliminary data, I hypothesize that DNA damage-triggered SUMO modification of key BER proteins is required to orchestrate a proper DNA damage response. To test my hypothesis, the ntg1∆SUMO mutant will be employed for the following Specific Aims: 1) to assess how SUMO modification impacts the repair capacity of Ntg1; 2) to explore connections between DNA damage-induced SUMO modification of Ntg1 and the DNA damage response pathway; and 3) to identify SUMO-dependent interactions with Ntg1 that could coordinate Ntg1 function with other cellular pathways. The proposed experiments seek to understand the role that Ntg1 plays in the DNA damage checkpoint and identify the sumoylation-dependent interactions required for this function. My long term goals are to understand the communication between BER and other DNA damage response pathways to provide insight into the mechanisms that regulate BER and possibly suggest new therapeutic targets for cancer.

 描述(申请人提供)：癌症是美国主要的死亡原因之一。基因不稳定性是癌症的一个标志，它促进了癌症的积累 越来越多的恶性肿瘤表型。为了防止遗传不稳定性，细胞发展了多种进化上保守的DNA损伤修复和DNA损伤反应途径，保护核和线粒体基因组。DNA损伤的一个主要原因是氧化性DNA损伤，它是由活性氧物种(ROS)引起的。DNA氧化损伤的主要修复途径是碱基切除修复(BER)途径，该途径从酵母到人类都是保守的。虽然BER途径在保护核和线粒体基因组方面发挥着重要作用，但这一途径是如何调控的尚不清楚。我们最近的研究通过关注酿酒酵母Ntg1来解决这一知识缺口，Ntg1是一种进化上保守的BER蛋白，可以识别和切除氧化碱基损伤。人类Ntg1的对应物Nthl1改变了与结肠癌和胃癌相关的功能/定位，突显了Nthl1在预防癌症和确定调节BER途径的机制方面的重要性。我们的工作表明，Ntg1和Nthl1都可以靶向细胞核或线粒体，以响应细胞器特异性的DNA损伤。与BER蛋白的翻译后修饰(S)有助于重新定位和/或调节的模型一致，我们在发芽酵母中的初步研究表明，在核氧化损伤后，Ntg1的核池被小泛素样修饰物(SUMO)修饰。重要的是，初步数据显示，人类Nthl1也可以被相扑修饰。为了评估这些BER蛋白相扑修饰的功能重要性，我们定位了Ntg1(K20，38,376,388,396)上的所有相扑修饰位点，并创建了一个不可相扑的(K&gt；R)NTG1变异体(NTG1∆相扑)。尽管重组NTG1DNA相扑在体外实验中保持了催化活性，但表达作为唯一拷贝的NTG1DNA相扑的细胞在∆损伤反应中表现出缺陷，未能阻止细胞周期对∆损伤的响应。基于这些初步数据，我推测，DNA损伤引发的关键BER蛋白的相扑修饰是协调适当的DNA损伤反应所必需的。为了验证我的假设，我们将使用NTG1∆相扑突变体来实现以下特定目的：1)评估相扑修饰如何影响NTG1的修复能力；2)探索DNA损伤诱导的NTG1相扑修饰与DNA损伤反应通路之间的联系；以及3)确定相扑依赖的与NTG1的相互作用，以协调NTG1与其他细胞通路的功能。拟议的实验试图了解Ntg1在DNA损伤检查点中扮演的角色，并确定这一功能所需的苏莫化依赖的相互作用。我的长期目标是了解BER和其他DNA损伤反应途径之间的联系，为调节BER的机制提供洞察力，并可能建议癌症的新治疗靶点。