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: 3157049
• 负责人: Muriel W Lambert
• 金额: $ 15.32万
• 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/3157049
• 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体细胞的分子机制 负责修复短波长引入 DNA 的损伤 紫外线（254 nm；UVC）光。 这个问题将使用两个方法来解决 我们开发的新的、经过验证的系统，用于 1) 纯化、表征、 并确定 DNA 核酸内切酶活性的作用位点，pI 7.6， 对 UVC 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，并且 2) 检查染色质结构在修饰该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以下疾病的患者的细胞： 色素性干皮病 (XP)，一种对阳光和皮肤显着敏感的疾病 紫外线和与阳光相关的皮肤变化的明显趋势， 包括许多癌症，在光照区域。 XP 培养细胞 患者对 UVC 光的敏感性显着增加，这在细胞中 大多数 XP 患者的缺陷与第一个核酸内切酶的缺陷有关 UVC 修复的核苷酸切除机制介导的步骤 光诱导的 DNA 损伤。 至少在最严重的一项 然而，有证据表明 XP (XPA) 的受影响的互补组 A 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，虽然 XP 可能是最好的 可用模型来研究阳光对人体皮肤的影响 由于暴露于环境因素（分子）而致癌 导致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 确定 UVC 核酸内切酶在 pI 7.6 时的活性实际上是 存在于 XPA 细胞中，但该核酸内切酶本身存在缺陷 或内切核酸酶所需的密切相关的辅因子 当 DNA 处于染色质形式时，其对 UVC 照射的 DNA 具有活性。 这 研究将更清楚地阐明染色质结构的精确作用 在此紫外核酸内切酶活性中。 我们独特的方法组合 应获得对负责的分子机制的宝贵见解 用于修复 UVC 光对 DNA 的损伤，并深入研究相关机制 紫外线对人体皮肤的影响。