DNA replication, DNA repair and microsatellite stability

描述(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。这项建议的目的是确定起稳定人类细胞微卫星序列作用的生化机制的全部补充。我们的工作假设是，细胞微卫星突变率是DNA复制过程中维持基因组稳定性的蛋白质的累积结果。我们已经开发了互补的体外/体外试验来研究报告微卫星在体细胞中的突变。特殊目的1将检验这样一种假设，即微卫星序列中DNA聚合酶的暂停可以阻碍复制分叉进程，并且RecQ解旋酶在微卫星DNA复制过程中具有特殊功能。通过不同序列的微卫星对复制中间产物进行生化分析，使用来自正常、Bloom和Werner综合征捐赠者的细胞系来实现BLM和WRN解旋酶的功能。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起到稳定微卫星DNA序列的作用。具体目标2将确定与复制分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的稳定性的贡献，并测试Mre11/NBS/Rad50复合体对人类细胞复制保真度的贡献。体外穿梭载体系统将用于自然产生的MLH1、PMS2、NBS1和hMre11缺陷的淋巴母细胞系，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hMre11的活动是否影响复制保真度。具体目标3将确定复制DNA聚合酶和Y家族DNA聚合酶对自发突变和微卫星稳定性的相对贡献。体外HSV-tk试验将用于分析DNA聚合酶增量和聚合酶kappa(Polkappa)在微卫星上的错误率。在体外实验中，我们将利用含有polkappa过表达载体或稳定的核酶来下调polkappa表达的细胞系来分析polkappa水平对自发细胞突变的影响。这些研究将确定对Polkappa活性的调节是否为旨在调节基因组稳定性的治疗干预的潜在途径。这项拟议的研究对模拟肿瘤进展具有直接意义，因为基因组监测机制的丧失将加速微卫星突变。微卫星等位基因长度可以直接影响基因表达。由于微卫星在人类群体中是多态的，这种对基因调控的影响可能是导致个体癌症风险的一个重要因素。

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.