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

 描述（由申请人提供）：癌症是美国的主要死亡原因之一。遗传不稳定性是一个促成癌症的标志， 恶性程度越来越高的癌症表型。为了防止遗传不稳定性，细胞已经发展了多种进化上保守的DNA损伤修复和DNA损伤反应途径，以保护核和线粒体基因组。DNA损伤的一个主要原因是由活性氧（ROS）引起的氧化性DNA损伤。氧化性DNA损伤的主要修复途径是碱基切除修复（BER）途径，其在酵母和人类中是保守的。尽管BER途径在保护核和线粒体基因组中起主要作用，但该途径如何调节尚不清楚。我们最近的研究通过关注S来解决这一知识缺口。酿酒酵母Ntg 1，一种进化上保守的BER蛋白，识别并切除氧化碱基损伤。人类Ntg 1对应物Nthl 1具有与结肠癌和胃癌相关的改变的功能/定位，突出了Nthl 1在预防癌症和定义调节BER途径的机制中的重要性。我们的工作表明，Ntg 1和Nthl 1都可以靶向细胞核或线粒体，以响应细胞器特异性DNA损伤。与BER蛋白的翻译后修饰有助于重新定位和/或调节的模型一致，我们在芽殖酵母中的初步研究表明，Ntg 1的核库在核氧化损伤后被小泛素样修饰剂（SUMO）修饰。重要的是，初步数据显示人Nthl 1也可以被SUMO修饰。为了评估这些BER蛋白的SUMO修饰的功能重要性，我们绘制了Ntg 1上的所有SUMO修饰位点（K20，38，376，388，396），并创建了一个不可SUMO化的（K->R）ntg 1变体（ntg 1 SUMO）。尽管重组ntg 1 α SUMO在体外试验中保留了催化活性，但表达ntg 1 α SUMO作为Ntg 1的唯一拷贝的细胞表现出DNA损伤应答的缺陷，不能阻止细胞周期对DNA损伤的应答。基于这些初步的数据，我假设，DNA损伤触发的SUMO修饰的关键BER蛋白是需要精心策划一个适当的DNA损伤反应。为了验证我的假设，ntg 1 SUMO突变体将用于以下特定目的：1）评估SUMO修饰如何影响Ntg 1的修复能力; 2）探索DNA损伤诱导的Ntg 1 SUMO修饰与DNA损伤反应途径之间的联系; 3）鉴定与Ntg 1的SUMO依赖性相互作用，从而协调Ntg 1与其他细胞途径的功能。拟议的实验旨在了解Ntg 1在DNA损伤检查点中发挥的作用，并确定此功能所需的sumoylation依赖性相互作用。我的长期目标是了解BER和其他DNA损伤反应途径之间的通信，以深入了解调节BER的机制，并可能提出新的癌症治疗靶点。

 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.