DNA replication, DNA repair and microsatellite stability

DNA 复制、DNA 修复和微卫星稳定性

• 批准号: 6993555
• 负责人: Kristin A Eckert
• 金额: $ 25.75万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6993555
• 关键词: DNA directed DNA polymerase; DNA repair; DNA replication; DNA replication origin; cell line; chemical stability; enzyme activity; gene expression; genetic polymorphism; genetic regulation; helicase; human tissue; neoplastic process; nucleic acid sequence; polymerase chain reaction; protein protein interaction; reporter genes; tissue /cell culture; two dimensional gel electrophoresis

DESCRIPTION (provided by applicant): The long-term goal of our research is to elucidate the role of microsatellite DNA sequence variation in neoplastic progression. The objective of this proposal is to identify the full complement of biochemical mechanisms that act to stabilize microsatellite sequences in human cells. Our working hypothesis is that cellular microsatellite mutation rates are the cumulative result of proteins acting to maintain genomic stability during DNA replication. We have developed complementary in vitro/ex vivo assays to study mutagenesis within reporter microsatellites in somatic human cells. Specific Aim 1 will test the hypothesis that DNA polymerase pausing within microsatellite sequences can impede replication fork progression, and that RecQ helicases have a specialized function during microsatellite DNA replication. Biochemical analyses of replication intermediates through microsatellites of differing sequence will be performed using cell lines from normal, Bloom and Werner syndrome donors to the function of BLM and WRN helicases. Mutation rates within the herpes simplex virus thymidine kinase (HSV-tk) gene reporter cassettes will be quantitated to determine whether these helicases function to stabilize microsatellite DNA sequences. Specific Aim 2 will determine the contribution of enzymatic activities associated with the replication fork in maintaining genome stability. We will test the contribution of mismatch repair proteins to the stability of tetranucleotide alleles and microsatellites with potential secondary structure, and test the contribution of the Mre11/NBS/Rad50 complex to human cell replication fidelity. The ex vivo shuttle vector system will be used in naturally occurring MLH1, PMS2, NBS1 and hMre11-defective lymphoblastoid cell lines, and in cells with gene expression down-regulated by antisense methods. Mutation rates and specificities will be determined to establish whether the activities of NBS and hMre11 affect replication fidelity. Specific Aim 3 will determine the relative contribution of replicative and Y family DNA polymerases to spontaneous mutagenesis and microsatellite stability. The in vitro HSV-tk assay will be used to analyze DNA polymerase delta and polymerase kappa (pol kappa) error rates at microsatellites. The effects of pol kappa levels on spontaneous cellular mutagenesis will be analyzed using the ex vivo assay cell lines containing either pol kappa overexpression vectors or stable ribozymes to down-regulated pol kappa expression. These studies will establish whether regulation of pol kappa activity is a potential avenue for therapeutic interventions aimed at regulating genome stability. This proposed research has direct implications for modeling tumor progression, as the loss of genomic surveillance mechanisms will accelerate microsatellite mutagenesis. Microsatellite allele lengths can directly affect gene expression. As microsatellites are polymorphic in human populations, this effect on gene regulation may be an important factor contributing to individual cancer risk.

描述（由申请人提供）：我们研究的长期目标是阐明微卫星DNA序列变异在肿瘤进展中的作用。本提案的目的是确定生物化学机制的完整补充，以稳定人类细胞中的微卫星序列。我们的工作假设是，细胞微卫星突变率是蛋白质的累积结果，以维持DNA复制过程中的基因组稳定性。我们已经开发了互补的体外/离体试验，以研究在人体细胞中的报告微卫星内的诱变。具体目标1将测试的假设，DNA聚合酶暂停在微卫星序列可以阻止复制叉的进展，RecQ解旋酶在微卫星DNA复制过程中有专门的功能。将使用来自正常、Bloom和Werner综合征供体的细胞系，通过不同序列的微卫星对复制中间体进行BLM和WRN解旋酶功能的生化分析。将对单纯疱疹病毒胸苷激酶（HSV-tk）基因报告盒内的突变率进行定量，以确定这些解旋酶是否具有稳定微卫星DNA序列的功能。具体目标2将确定与复制叉相关的酶活性在维持基因组稳定性方面的贡献。我们将测试错配修复蛋白对四核苷酸等位基因和具有潜在二级结构的微卫星稳定性的贡献，并测试Mre 11/NBS/Rad 50复合物对人类细胞复制保真度的贡献。离体穿梭载体系统将用于天然存在的MLH 1、PMS 2、NBS 1和hMre 11缺陷型淋巴母细胞样细胞系，以及通过反义方法下调基因表达的细胞。将确定突变率和特异性以确定NBS和hMre 11的活性是否影响复制保真度。具体目标3将确定复制型和Y家族DNA聚合酶对自发诱变和微卫星稳定性的相对贡献。体外HSV-tk试验将用于分析微卫星的DNA聚合酶δ和聚合酶κ（pol kappa）错误率。将使用含有pol κ过表达载体或稳定核酶以下调pol κ表达的离体试验细胞系分析pol κ水平对自发细胞诱变的影响。这些研究将确定pol kappa活性的调节是否是旨在调节基因组稳定性的治疗干预的潜在途径。这项拟议的研究对建模肿瘤进展有直接影响，因为基因组监视机制的丧失将加速微卫星诱变。微卫星等位基因的长度可以直接影响基因的表达。由于微卫星在人群中具有多态性，这种对基因调控的影响可能是导致个体癌症风险的重要因素。