ROLE OF ISOLATED NUCLEAR PROTEINS IN DNA REPAIR

分离的核蛋白在 DNA 修复中的作用

• 批准号: 3157053
• 负责人: Muriel W Lambert
• 金额: $ 13.79万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-30 至 1990-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3157053
• 关键词: endonuclease; human subject; light adverse effect; lymphoblast; nucleoproteins; nucleosomes; skin neoplasms; solar radiation; xeroderma pigmentosum

Our objective is to elucidate the molecular mechanisms in human cells responsible for repair of lesions introduced into DNA by short wavelength ultraviolet (254 nm; UVC) light. This problem will be approached using two new, proven systems, which we have developed, to 1) purify, characterize, and determine the site(s) of action of a DNA endonuclease activity, pI 7.6, with increased activity on UVC damaged DNA, which we have already isolated and partially purified from human lymphoblastoid cell chromatin, and to 2) examine the role of chromatin structure in modifying this endonuclease activity, using a reconstituted nucleosomal substrate. Normal human lymphoblastoid cells will be used as well as those from patients with xeroderma pigmentosum (XP), a disorder with marked sensitivity to sun and UV light and a marked tendency to develop sun related skin changes, including numerous cancers, in light exposed areas. Cultured cells from XP patients show markedly increased sensititivy to UVC light, which, in cells from most XP patients is related to a defece in the first, endonuclease mediated, step of the nucleotide excision mechanism for repair of UVC light-induced lesions in DNA. In at least one of the most severely affected complementation groups, A, of XP (XPA), however, there is evidence that the defect is not in the ability of the UV endonuclease to incise DNA when it is in the form of chromatin. Thus, although XP is perhaps the best available model for the effects of sunlight on human skin and for carcinogenesis due to exposure to an environmental agent, the molecular mechanisms responsible for this defect have remained obscure. Our approach should allow us to elucidate these mechanisms. It has already enabled us to ascertain that the UVC endonuclease activity at pI 7.6 is, in fact, present in XPA cells but that there is a defect in this endonuclease itself or in a closely associated cofactor which is needed for endonuclease activity on UVC irradiated DNA when it is in the form of chromatin. The studies will more clearly elucidate the precise role of chromatin structure in this UV endonuclease activity. Our unique combination of approaches should obtain valuable insight into the molecular mechanisms responsible for repair of UVC light damage to DNA, and into the mechanisms responsible for UV light effects on human skin.

我们的目标是阐明人类细胞中的分子机制。 负责DNA短波导入损伤的修复 紫外线(254 nm；UVC)光。这个问题将使用两个 我们开发的新的、经过验证的系统，用于1)提纯、表征、 并确定了DNA内切酶活性的作用部位(S)，等电点7.6， 随着紫外线损伤DNA的活性增加，我们已经分离出 并从人淋巴母细胞染色质中部分纯化，并至2) 研究染色质结构在修饰该核酸内切酶中的作用 活性，使用重组的核小体底物。正常人 淋巴母细胞将被使用，以及来自于 着色性干皮病(XP)，一种对阳光和 紫外线和明显的与阳光有关的皮肤变化的趋势， 包括许多癌症，在光线暴露的地区。来自XP的培养细胞 患者对UVC光的敏感度显著增加，在细胞中 大多数XP患者的症状与第一种核酸内切酶缺陷有关 修复UVC的核苷酸切除机制的介导性步骤 光诱导的DNA损伤。在至少一个最严重的 然而，有证据表明，XP(XPA)的受影响互补群A 缺陷不在于紫外线核酸内切酶切割DNA的能力 当它以染色质的形式出现时。因此，尽管XP可能是最好的 太阳光对人体皮肤影响的可用模型 由于暴露在环境介质分子中而致癌 造成这一缺陷的机制仍然不清楚。我们的方法 应该使我们能够阐明这些机制。它已经使我们能够 为了确定等电点7.6的UVC内切酶活性实际上是， 在XPA细胞中存在，但这种内切酶本身存在缺陷 或在核酸内切酶所需的密切相关的辅因子中 当DNA以染色质形式存在时，紫外线照射的DNA具有活性。这个 研究将更清楚地阐明染色质结构的确切作用 在这种紫外线核酸内切酶的活性。我们独特的方法组合 应该对相关的分子机制有价值的了解 用于修复紫外线对DNA的光损伤，并进入负责的机制 紫外线对人体皮肤的影响。