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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

基本信息

批准号：
6908109
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
项目类别：
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/6908109
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

项目摘要

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）：着色性干皮病 (XP) 是一种疾病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、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 到 G 代表阳光（UVB）损伤的核苷酸切除修复（NER）缺陷 XPV 代表皮肤 DNA 复制缺陷脱氧核糖核酸。 XP 细胞均表现出紫外线诱导的突变升高，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，D' 类聚合酶同源大肠杆菌，以无差错的方式发挥作用，防止紫外线损伤。该酶是一种即使在未受损的 DNA 上也具有高错误率的分布式聚合酶。我们有将基因映射到 6p2l，识别出 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 患者中被删除。我们观察到启动子有多个 AP1 和 SP1 位点，表明损伤响应调节。我们发现了组成型启动子的表达具有毒性，但已实现多种 XPV 表型的功能校正（紫外线存活、细胞凋亡）使用融合蛋白的表达。我们还有发现 XPV 细胞中紫外线损伤后 DNA 复制涉及双重复制链修复/重组系统涉及 hMre1 1/hRad50/Nbs1 并取决于第 53 页。了解具有如此高错误率的聚合酶如何有助于紫外线损伤 DNA 的无差错复制，以及如何对其进行调节以避免猖獗的错误产生和毒性是非常重要的，也是重点这个项目的。我们的具体目标是：目标 I：通过以下方式了解机制：其中低保真 hRad3O 聚合酶受到调节以维持遗传在正常和转化细胞中的稳定性。我们将设计表达载体在体外补充 XPV 表型，并使用它们来识别结合控制和紫外线损伤细胞的合作伙伴。目标二：了解角色的作用 hRad3O 缺陷细胞中 S 期检查点的重组。我们将确定 hMre 11 重组在 S 特定阶段的作用期并识别 S 期信号转导途径的组成部分。目的 III：培育hRad3O功能缺陷的小鼠品系。我们将表达 hRad3O 位于角蛋白 14 启动子上，可在皮肤中过度表达，并使体内靶向敲除 mRad3O，以确定 hRad3O 在促进并预防癌变。

项目成果

期刊论文数量（21）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Increased ultraviolet sensitivity and chromosomal instability related to P53 function in the xeroderma pigmentosum variant.

DOI：
发表时间：
1999-03
期刊：
Cancer research
影响因子：
11.2
作者：
J. Cleaver;V. Afzal;L. Feeney;M. Mcdowell;W. Sadinski;J. Volpe;D. Busch;D. Coleman;D. W. Ziffer;Y. Yu;H. Nagasawa;J. Little
通讯作者：
J. Cleaver;V. Afzal;L. Feeney;M. Mcdowell;W. Sadinski;J. Volpe;D. Busch;D. Coleman;D. W. Ziffer;Y. Yu;H. Nagasawa;J. Little

DNA replication arrest in XP variant cells after UV exposure is diverted into an Mre11-dependent recombination pathway by the kinase inhibitor wortmannin.

紫外线照射后，XP 变异细胞中的 DNA 复制停滞被激酶抑制剂渥曼青霉素转入依赖于 Mre11 的重组途径。