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（由申请人提供）：Xeroderma色素（XP）是一种疾病 遗传缺乏使患者容易受到阳光诱发的癌症 皮肤，包括鳞状和基底细胞癌和黑色素瘤。 XP是一个 涉及至少8个基因的多基因疾病：XP组A至G表示 阳光（UVB）核苷酸切除修复（NER）的缺陷损害 皮肤的DNA，XPV表示损坏的复制不足 脱氧核糖核酸。 XP细胞均显示出较高的紫外线诱导的诱变，这与 阳光诱发癌症的风险增加。 XPV基因编码DNA 聚合酶，hrad3o，po1 n，与Umuc同源，D'类别聚合酶 e.coli，并在无误途径中起作用紫外线损伤。酶是 甚至在未损坏的DNA上具有高误差率的分布聚合酶。我们有 将基因映射到6P2L，确定了10个编码外显子和一个未翻译的外显子 I.外显子II在某些正常转录本和XP患者中被删除。我们 观察启动子具有多个AP1和SP1位点，这表明 损害响应性调节。我们从组成型启动子发现表达 有毒，但已经实现了几种XPV表型的功能校正 （紫外线存活率，凋亡）使用融合蛋白的表达。我们也有 发现XPV细胞中紫外线损伤后的DNA复制涉及双重 涉及HMRE1 1/hrad50/nbs1的链修复/重组系统，取决于 p53。了解具有如此高误差的聚合酶如何贡献 紫外线损坏的DNA的无错误复制，以及如何调节它以避免 猖ram的错误产生和毒性至关重要，重点是 这个项目。我们的具体目的是：目标：了解 低保真度HRAD3O聚合酶受调节以维持遗传 正常细胞和转化细胞的稳定性。我们将设计表达向量 在体外补充XPV表型，并使用这些表型识别结合 对照和紫外线损坏的细胞的伴侣。目标II：了解 在HRAD3O缺乏的细胞中S相检查点中的重组。我们 将确定HMRE 11重组在S的特定阶段的作用 相位并确定S相信号转导途径的组件。目的 III：在HRAD3O功能中发展小鼠菌株。我们将表达 角蛋白14启动子上的hrad3o在皮肤中过度表达，并制作 MRAD3O在体内的靶向敲除，以识别HRAD3O在促进中的作用 并防止致癌。