The XP Variant: A Human Mutator Gene for UV Damage

描述(申请人提供)：着色性干皮病(XP)是一种 遗传缺陷使患者容易患上阳光诱发的癌症 皮肤，包括鳞状细胞癌和基底细胞癌以及黑色素瘤。XP是一种 至少涉及8个基因的多基因疾病：XP组A至G代表 日光(UVB)损伤的核苷酸切除修复(NER)缺陷 皮肤的DNA，而XPV代表着受损的 DNAXP细胞都表现出紫外线诱导的高诱变性，这与 阳光引发癌症的风险增加。XPV基因编码一种DNA 聚合酶，hRad3O，PO1n，与UMUC，D‘类聚合酶同源 E.Coli，并在紫外线损伤的无错误途径中发挥作用。这种酶是一种 分布聚合酶，即使对未受损的DNA也有很高的错误率。我们有 将该基因定位于6p21，确定了10个编码外显子和一个未翻译的外显子 一、外显子II在一些正常转录本和一名XP患者中缺失。我们 观察到启动子有多个AP1和SP1位点，表明 损害反应法规。我们发现了一个构成启动子的表达 是有毒的，但已经实现了对几种XPV表型的功能纠正 (紫外线存活，细胞凋亡)使用融合蛋白表达。我们还有 发现紫外线损伤后XPV细胞中的DNA复制涉及双倍 涉及hMre1 1/hRad50/Nbs1的链修复/重组系统 P53。理解具有如此高错误率的聚合酶如何有助于 紫外线损伤DNA的无错误复制，以及如何对其进行监管以避免 猖獗的错误产生和毒性是主要的重要性和重点 这个项目的。我们的具体目标是：目标一：通过 低保真的hRad3O聚合酶被调控以维持遗传 正常细胞和转化细胞的稳定性。我们将设计表达载体 在体外补充XPV表型，并用这些来鉴定结合 控制和紫外线破坏细胞的合作伙伴。目标二：了解 缺失hRad30的细胞在S期检查点(S)的重组。我们 将决定hMRE11重组在S特定阶段中的作用 并确定S时相信号转导通路的组成成分。目标 III：建立hRad3O功能缺陷小鼠品系。我们会表示 HRad3O对角蛋白14启动子在皮肤中的过度表达，并使 体内靶向敲除mRad3O以确定hRad3O在促进 和预防癌症的发生。

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.