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。取决于暴露，UV诱导的损伤例如（6-4）嘧啶-嘧啶酮光产物（6- 4PP）和环丁烷嘧啶二聚体（CPD）可通过UV的DNA吸收形成。如果这些病变没有得到适当的修复，它们可能会导致DNA受损，突变和染色体畸变。更具体地说，由于修复途径的问题，紫外线诱导的损伤的形成和可能的保留可能有助于皮肤癌的发展。负责修复UV诱导的损伤的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，在细胞内具有许多功能，特别是其在DNA修复途径中的作用。在DNA损伤时，它被激活并能够形成有助于修复的聚（ADP-核糖）部分（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修复了由于氧化、脱氨基或烷基化而形成的小的非螺旋扭曲损伤。有几篇论文指出它与途径中的蛋白质（即XRCC 1，DNA聚合酶2和DNA连接酶III）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导致嘧啶二聚体和其他光损伤的保留，我们在人类角质形成细胞中发现了类似的结果。虽然支持PARP-1参与NER的数据正在增加，但其作用机制尚未完全阐明。有几种蛋白质参与NER的早期步骤，值得注意的是XPC-hHR 23 B，DDB 1和XPA。特别是，XPA已被证明含有pADPr结合基序，并已检查其对pADPr的亲和力。我们的假设是，PARP-1在紫外线诱导的DNA损伤的修复中起作用，此外，其激活和随后形成的pADPr部分允许其与NER中的必需蛋白质相互作用。我们将通过1）使用体外和体内系统确定PARP-1缺陷是否改变角质形成细胞中紫外线诱导的DNA损伤的修复，2）鉴定PARP-1的活化是否将导致pADPr亚基的形成，所述pADPr亚基充当NER途径的早期组分如XPA的支架，以及3）确定PARP-1的什么效应损失。1活性对早期NER组分的影响。我们的初步研究结果表明，XPA是与pADPr部分和免疫沉淀与PARP-1紫外线照射后，支持的假设，pADPr可能是作为一个支架的NER的早期组件。获得关于PARP-1在修复UV诱导的病变中的作用的信息将拓宽我们对NER的理解，这可能导致改进的方法来防止皮肤中引起病变的癌症的保留。 公共卫生相关性：NIEHS战略计划特别指出：“对改变DNA损伤、修复和维护的环境因素的研究是一个重要的研究领域，特别是在衰老、癌症和细胞死亡方面。“紫外线是导致皮肤癌的主要环境因素，该项目旨在从分子水平上了解PARP在DNA修复活动中的作用。

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.