Novel deubiquitinating activities targeting DNA damage recognition in NER

 描述(申请人提供)：许多人类疾病是由基因组和环境因子之间复杂的相互作用引起的。突变是未修复的DNA损伤的常见后果。核苷酸切除修复(NER)通路在环境诱变剂诱导的DNA损伤修复中起着特别重要的作用。NER修复由紫外线辐射、顺铂或活性氧物种(ROS)等损害剂以及香烟烟雾中发现的许多化学物质造成的各种扭曲螺旋的“笨重”DNA损伤。NER有两个子途径，称为整体基因组修复(GG-NER)和转录偶联修复(TC-NER)。这些途径的主要不同之处在于对损害的识别。被病变阻断的RNA聚合酶启动TC-NER，将其限制在活性基因的转录链上，而GG-NER则调查整个基因组以寻找扭曲的DNA损伤。因此，GG-NER需要特定的蛋白质来识别损伤。着色性干皮病互补组C(XPC)和着色性干皮病互补组E(XPE)是GG-NER中识别DNA损伤的两个重要损伤识别因子。泛素化在TC-NER和GG-NER中都起着关键作用。在GG-NER过程中，DDB2和XPC都被泛素化共价修饰。尽管泛素化在DDB2和XPC对DNA损伤识别的调控中起着至关重要的作用，但泛素在DDB2和XPC中的结合位置和泛素链拓扑结构仍未确定。需要这些信息来理解为什么泛素化的XPC是稳定的，去泛素化后XPC可以被回收，而泛素化的DDB2是不稳定的，被蛋白酶体降解。顺便说一句，我们的初步研究表明，两个新的去泛素化酶(DUBS)，USP24和OTUD4，在没有紫外线照射的情况下，对XPC和DDB2的稳态水平进行不同的调节。紫外线照射后的时间进程实验也表明，紫外线照射后OTUD4耗竭细胞中XPC泛素化水平增加。由于XPC和DDB2在DNA损伤识别中发挥作用，我们假设USP24和OTUD4调节NER。本研究的主要目的是确定DDB2和XPC中的泛素连接位点，揭示决定XPC和DDB2“命运”的泛素连锁拓扑结构，并确定USP24和OTUD4通过其去泛素酶活性调节XPC和DDB2中的机制。我们的长期目标是了解DNA损伤是如何在人类细胞中识别和修复的。关于XPC和DDB2泛素化和去泛素化的知识将有助于理解DNA损伤识别的动力学。R21资助机制将使我们能够定义XPC和DDB2泛素化的性质，并研究OTUD4和USP24在DNA修复中的新角色。为了实现我们的目标，我们提出了以下三个具体目标。在目标1中，我们将确定紫外线照射前后XPC/DDB2泛素化的附着位置和性质。目标2将定义USP24和OTUD4控制XPC和DDB2值的机制。在目标3中，我们将确定USP24和OTUD4在DNA修复和基因组稳定性中的生理作用。

 DESCRIPTION (provided by applicant): Many human diseases result from complex interactions between genome and environmental agents. Mutation is a frequent consequence of unrepaired DNA damage. The nucleotide excision repair (NER) pathway plays a particularly important role in the repair of environmental mutagen-induced DNA damage. NER repairs a wide variety of helix-distorting `bulky' DNA lesions that result from damaging agents such as UV radiation, cisplatin or reactive oxygen species (ROS), as well as many chemicals found in cigarette smoke. There are two sub- pathways of NER, termed global genomic repair (GG-NER) and transcription-coupled repair (TC-NER). These pathways differ mainly in their recognition of damage. While an RNA polymerase blocked by a lesion initiates TC-NER, restricting it to the transcribed strand of active genes, GG-NER surveys the entire genome for distorting DNA lesions. Therefore, GG-NER requires specific proteins for damage recognition. Xeroderma pigmentosum complementation group C (XPC) and Xeroderma pigmentosum complementation group E (XPE or DDB2) are two important damage recognition factors that recognize DNA lesions during GG-NER. Ubiquitination plays crucial roles in both TC-NER and GG-NER. DDB2 and XPC are both covalently modified by ubiquitination during GG-NER. Although ubiquitination plays a crucial role in the regulation of DNA damage recognition by DDB2 and XPC, the sites of ubiquitin attachment in DDB2 and XPC and ubiquitin chain topologies remain unidentified. These pieces of information are needed to understand why ubiquitinated XPC is stable and XPC can be recycled after deubiquitination, while ubiquitinated DDB2 is labile and degraded by the proteasome. Incidentally, our preliminary studies show that two novel deubiquitinating enzymes (DUBs), USP24 and OTUD4, differentially regulate the steady state levels of XPC and DDB2 in the absence of UV irradiation. Time course experiments following UV irradiation also show increased levels of XPC ubiquitination after UV irradiation in OTUD4 depleted cells. Since XPC and DDB2 play roles in DNA damage recognition, we hypothesize that USP24 and OTUD4 regulate NER. The main objective of this study is to identify ubiquitin attachment sites in DDB2 and XPC, reveal ubiquitin linkage topologies that decide the "fate" of XPC and DDB2 and define the mechanisms by which USP24 and OTUD4 regulate XPC and DDB2 through their deubiquitinase activities. Our long-term goal is to understand how DNA damage is recognized and repaired in human cells. Knowledge about XPC and DDB2 ubiquitination and deubiquitination will help understand the dynamics of DNA damage recognition. The R21 funding mechanism will allow us to define the nature of XPC and DDB2 ubiquitination and investigate the novel roles of OTUD4 and USP24 in DNA repair. The following three Specific Aims are proposed to achieve our goal. In Aim 1, we will identify attachment sites and nature of XPC/DDB2 ubiquitination before and after UV irradiation. Aim 2 will define the mechanisms by which USP24 and OTUD4 control the levels of XPC and DDB2. In Aim 3 we will determine the physiological roles of USP24 and OTUD4 in DNA repair and genomic stability.