Role of poly (ADP-ribose)in the Repair of Ultraviolet Induced DNA Damage

描述（由申请人提供）：我们每天都暴露在紫外线（UV）辐射下。根据暴露的不同，紫外线诱导的损伤，如（6-4）嘧啶-嘧啶光产物（6-4 PPs）和环丁烷嘧啶二聚体（CPDs），可以通过DNA吸收紫外线形成。如果这些损伤没有得到适当的修复，就会导致DNA受损、突变和染色体畸变。更具体地说，由于修复途径中的问题，紫外线诱导的病变的形成和可能的保留可能有助于皮肤癌的发展。紫外线诱导损伤的修复途径是核苷酸切除修复（NER）。聚（ADP-核糖）聚合酶-1 （PARP-1）是一种锌指蛋白，在细胞内具有多种功能，特别是在DNA修复途径中发挥作用。当DNA受到损伤时，它被激活并能够形成辅助修复的多聚（ADP-核糖）片段（pADPr）。PARP-1在碱基切除修复（BER）中的作用已经得到了很好的证实，BER可以修复由于氧化、脱氨或烷基化而形成的小的、非螺旋扭曲的损伤。一些论文已经注意到它与通路内的蛋白质（即XRCC1， DNA聚合酶2和DNA连接酶III）的直接相互作用。关于NER，越来越多的证据表明PARP-1可能在这一途径中发挥作用。最近的研究表明，抑制PARP会导致嘧啶二聚体和其他光斑的保留，我们在人角质形成细胞中发现了类似的结果。虽然支持PARP-1参与NER的数据越来越多，但其作用机制尚未得到充分阐明。有几种蛋白质参与了NER的早期步骤，值得注意的是XPC-hHR23B， DDB1和XPA。特别是，XPA已被证明含有一个pADPr结合基序，并且其对pADPr的亲和力已被研究。我们的假设是，PARP-1在紫外线诱导的DNA损伤修复中发挥作用，此外，它的激活和随后形成的pADPr片段允许它与NER中必需的蛋白质相互作用。我们将通过以下方法来验证这一假设：1)在体外和体内系统中确定PARP-1缺乏是否会改变角质形成细胞中紫外线诱导的DNA损伤的修复；2)确定PARP-1的激活是否会导致pADPr亚基的形成，这些亚基作为NER通路早期组分（如XPA）的支架；3)使用分子和生化方法确定PARP-1活性丧失对早期NER组分的影响。我们的初步研究结果表明，XPA与pADPr片段相关，并且在紫外线照射后与PARP-1发生免疫沉淀，这支持了pADPr可能作为NER早期成分支架的假设。获得关于PARP-1在紫外线诱导的病变修复中的作用的信息将拓宽我们对NER的理解，这可能会导致改进预防皮肤中致癌病变保留的方法。

DESCRIPTION (provided by applicant): We are exposed to ultraviolet (UV) radiation on a daily basis. Depending on the exposure, UV-induced lesions such as (6-4)pyrimidine-pyrimidone photoproducts (6-4 PPs) and cyclobutane pyrimidine dimers (CPDs), can be formed by DNA absorption of UV. If such lesions are not properly repaired they can lead to damaged DNA, mutations and chromosome aberrations. More specifically, the formation and possible retention of UV-induced lesions due to problems in repair pathways could contribute to the development of skin cancer. The pathway responsible for the repair of UV-induced lesions is nucleotide excision repair (NER). Poly(ADP- ribose)polymerase-1 (PARP-1) is a zinc-finger protein with many functions within the cell, one in particular being its role in DNA repair pathways. Upon DNA damage it becomes activated and is able to form poly(ADP- ribose) moieties (pADPr) that assist in repair. PARP-1's role in base excision repair (BER), which repairs small, non-helix distorting lesions formed as a result of oxidation, deamination or alkylation, has been well established. Several papers have noted its direct interaction with proteins within the pathway (i.e. XRCC1, DNA polymerase 2, and DNA Ligase III). With regard to NER, there is growing evidence that PARP-1 may have a role in this pathway. Recent studies suggest that inhibition of PARP leads to the retention of pyrimidine dimers and other photolesions and we find similar results in human keratinocytes. While the data supporting PARP-1's involvement in NER is increasing, the mechanism of action has yet to be fully elucidated. There are several proteins that are involved in the early steps of NER, of note are XPC-hHR23B, DDB1 and XPA. In particular, XPA has been shown to contain a pADPr binding motif and its affinity for pADPr has been examined. Our hypothesis is that PARP-1 plays a role in the repair of ultraviolet induced DNA damage, and furthermore, its activation and subsequent formation of pADPr moieties allow it to interact with essential proteins in NER. We will test this hypothesis by 1) establishing whether PARP-1 deficiency alters the repair of ultraviolet induced DNA damage in keratinocytes using in vitro and in vivo systems, 2) identifying whether activation of PARP-1 will lead to the formation of pADPr subunits which act as a scaffold for early components of the NER pathway such as XPA and 3) establishing what effect loss of PARP-1 activity has on early NER components using molecular and biochemical approaches. Our preliminary findings showed that XPA is associated with pADPr moieties and immunoprecipitated with PARP-1 following UV exposure, supporting the hypothesis that pADPr may be acting as a scaffold for early components of NER. Obtaining information about PARP-1's role in the repair if UV-induced lesions will broaden our understanding of NER which may result in improved methods to prevent the retention of cancer causing lesions in the skin. PUBLIC HEALTH RELEVANCE: The NIEHS Strategic Plan specifically notes "the study of environmental factors that modify DNA damage, repair, and maintenance is an important area of investigation, particularly with regard to aging, cancer, and cell death." UVR is the major environmental agent contributing to skin cancer and this project is aimed at gaining a molecular understanding of PARP's role in DNA repair activities.