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: 3157052
• 负责人: Muriel W Lambert
• 金额: $ 13.96万
• 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/3157052
• 关键词: endonuclease; human subject; light adverse effect; lymphoblast; 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 负责修复由短波长引入DNA的损伤 紫外（254 nm; UVC）光。 这个问题将使用两个 新的，经过验证的系统，我们已经开发，1）纯化，表征， 并确定DNA内切酶活性的作用位点，pI 7.6， 紫外线损伤DNA的活性增加，我们已经分离出了这些DNA 并从人淋巴母细胞样细胞染色质中部分纯化，和2） 检查染色质结构在修饰这种核酸内切酶中的作用 活性，使用重构的核小体底物。 正常人 将使用淋巴母细胞以及来自患有 着色性干皮病（XP），一种对阳光明显敏感的疾病， 紫外线和明显的趋势，发展太阳相关的皮肤变化， 包括许多癌症，在光暴露区域。 来自XP的培养细胞 患者对UVC光的敏感性显著增加， 大多数XP患者的这种缺陷与第一种核酸内切酶的缺陷有关， 介导的，核苷酸切除机制的步骤，用于修复UVC 光诱导的DNA损伤 在至少一个最严重的 受影响的互补组，A，XP（XPA），然而，有证据表明， 缺陷不在于UV核酸内切酶切割DNA的能力 当它以染色质的形式存在时。 因此，尽管XP可能是最好的 太阳光对人体皮肤的影响以及 致癌作用，由于暴露于环境因素，分子 造成这种缺陷的机制仍然不清楚。 我们的方法 应该能让我们阐明这些机制 它已经使我们能够 为了确定在pI7.6的UVC核酸内切酶活性，事实上， 存在于XPA细胞中，但这种内切核酸酶本身存在缺陷 或在核酸内切酶所需的密切相关的辅因子中 当UVC照射的DNA处于染色质形式时，对UVC照射的DNA的活性。 的 研究将更清楚地阐明染色质结构的确切作用 在此UV核酸内切酶活性。 我们独特的方法组合 应该可以获得有价值的深入了解的分子机制负责 用于修复UVC光对DNA的损伤，并进入负责的机制 紫外线对人体皮肤的影响。