The XP Variant: A Human Mutator Gene for UV Damage

XP 变体：导致紫外线损伤的人类突变基因

• 批准号: 6769587
• 负责人: JAMES E CLEAVER
• 金额: $ 33.19万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6769587
• 关键词: DNA damage; DNA repair; biological signal transduction; gene targeting; genetic promoter element; genetic strain; genetically modified animals; laboratory mouse; neoplasm /cancer genetics; nucleic acid sequence; radiation carcinogenesis; transfection /expression vector; ultraviolet radiation; xeroderma pigmentosum

DESCRIPTION (Provided by Applicant): Xeroderma pigmentosum (XP) is a disease in which a genetic deficiency predisposes patients to sunlight induced cancers of the skin, including squamous and basal cell cancers and melanomas. XP is a multigenic disease involving at least 8 genes: XP groups A through G represent deficiencies in nucleotide excision repair (NER) of sunlight (UVB) damage to the DNA of the skin, and XPV represents a deficiency in replication of damaged DNA. XP cells all show elevated UV-induced mutagenesis, which correlates with the increased risk for sunlight induced cancer. The XPV gene encodes a DNA polymerase, hRad3O, po1 n, homologous to the UMUC, D' class of polymerases in E.coli, and functions in an error-free pathway for UV damage. The enzyme is a distributive polymerase with a high error-rate on even undamaged DNA. We have mapped the gene to 6p2l, identified 10 coding exons, and an untranslated exon I. Exon II is deleted in some normal transcripts and in an XP patient. We observe that the promoter has multiple AP1 and SP1 sites, suggesting a damage-responsive regulation. We found expression from a constitutive promoter to be toxic, but have achieved functional correction of several XPV phenotypes (UV survival, apoptosis) using expression of a fusion protein. We have also found that DNA replication after UV damage in XPV cells involves a double strand repair/recombination system involving hMre1 1/hRad50/Nbs1 and depends on p53. Understanding how a polymerase with such a high error-rate contributes to error-free replication of UV damaged DNA, and how it is regulated to avoid rampant error generation and toxicity is of major importance and the emphasis of this project. Our specific aims are: Aim I: To understand the mechanisms by which the low fidelity hRad3O polymerase is regulated to maintain genetic stability in normal and transformed cells. We will design expression vectors that complement XPV phenotypes in vitro, and use these to identify binding partners in control and UV damaged cells. Aim II: To understand the role of recombination in the S phase checkpoint(s) in cells deficient in hRad3O. We will determine the role of hMre 11 recombination in specific stages of the S phase and identify components of the S phase signal transduction pathways. Aim III: To develop mouse strains defective in hRad3O functions. We will express hRad3O on the keratin 14 promoter for over-expression in the skin, and make targeted knockout of mRad3O in vivo to identify roles for hRad3O in promoting and preventing carcinogenesis.

描述（申请人提供）：着色性干皮病（XP）是一种 这种基因缺陷使患者容易患上阳光诱发的癌症， 皮肤癌，包括鳞状细胞癌、基底细胞癌和黑色素瘤。XP是一 涉及至少8个基因的多基因疾病：XP组A至G代表 在核苷酸切除修复（NER）的太阳光（UVB）损伤的缺陷， 皮肤的DNA，而XPV代表了受损DNA复制的缺陷。 DNA. XP细胞都显示出升高的UV诱导的诱变，这与 增加阳光诱发癌症的风险。XPV基因编码一种DNA 聚合酶，hRad 30，po 1 n，与UMUC，D'类聚合酶同源， 大肠杆菌，并在紫外线损伤的无错误途径中发挥作用。述酶是 分布式聚合酶即使在未受损的DNA上也具有高错误率。我们有 将该基因定位于6p 2l，鉴定了10个编码外显子和一个非翻译外显子 I.外显子II在一些正常转录本和XP患者中缺失。我们 观察到启动子有多个AP 1和SP1位点，表明 损害响应监管。我们发现从组成型启动子表达 是有毒的，但已经实现了几个XPV表型的功能校正 (UV存活、凋亡）。我们还 发现XPV细胞中紫外线损伤后的DNA复制涉及双链断裂， 涉及hMre 11/hRad 50/Nbs 1的链修复/重组系统，并依赖于 第53页。了解具有如此高错误率的聚合酶如何有助于 UV损伤DNA的无错误复制，以及如何调节以避免 猖獗的错误生成和毒性是非常重要的， of this project项目.我们的具体目标是：目标一：了解机制， 其中低保真度hRad 3 O聚合酶被调节以维持遗传稳定性， 在正常和转化细胞中的稳定性。我们将设计表达载体 其在体外补充XPV表型，并使用这些来鉴定结合 对照组和紫外线损伤组的细胞中的伴侣。目标二：了解 在缺乏hRad 3 O的细胞中，S期检查点的重组。我们 将确定hMre 11重组在S 阶段，并确定S期信号转导途径的组成部分。目的 III：开发hRad 3 O功能缺陷的小鼠品系。我们将表达 hRad 3 O对角蛋白14的启动子在皮肤中过度表达， 体内靶向敲除mRad 3 O以鉴定hRad 3 O在促进 和预防致癌作用。