MUTATIONAL MECHANISMS OF REPETITIVE DNA IN HUMAN CELLS

本研究的目的是评估 短串联重复序列自发突变和致癌物诱导突变 (STR)人类细胞中的DNA位点。 我们建议利用互补性 体外/离体诱变试验，以检验DNA 聚合酶错误是STR位点突变的潜在来源 体内基因座。 含有STR基序和 将通过插入二核苷酸构建独特的序列基序， 四核苷酸STR序列在5个素数编码的框架内 单纯疱疹病毒胸苷激酶（HSV-tk）基因的区域。 构成ATP结合位点的DNA序列构成了 唯一的DNA对照，以直接比较 STR基序的错误。 这些目标将被用作DNA模板 在人DNA聚合酶催化的体外DNA合成过程中， β和小牛胸腺聚合酶α-引发酶复合物。 的 将对产生的突变进行分析， 两个基序中聚合酶介导的错误的频率， 确定STR序列中精确的体外聚合酶错误率， 重复大小和碱基组成的函数。 双峰目标 序列也将被掺入Ori-Rho-TK穿梭载体中， 在人淋巴母细胞中游离复制。 突变 在定义的穿梭载体复制过程中产生， 培养物中前导链和滞后链DNA复制的条件 将分析正常细胞以确定定量突变率 STR基因座作为序列组成的函数。 双峰 穿梭载体将用于证明DNA加合物是否 由两种不同的致癌物产生，芳基胺化剂N- 苯甲酰氧基-N-甲基-4-氨基偶氮苯和烷基化剂N- 乙基-N-亚硝基脲可诱导STR基序突变。 两 将遵循突变诱导的途径，获得/丧失 连续重复序列中整数重复单元与碱基替换 不连续重复序列中的突变。 由此产生的数据 建议将：一）加强我们对机制的理解， 通过提供突变定量的人类体细胞诱变 重复DNA的比率，和II）建立重复DNA的程度， DNA被化学致癌物破坏。 我们的长期研究 目的是检验重复DNA中的突变 提供了基因型变异的重要来源， 肿瘤进展

The goal of this research proposal is to evaluate the significance of spontaneous and carcinogen-induced mutations at short tandem repeat (STR) DNA loci in human cells. We propose to utilize complementary in vitro/ex vivo mutagenesis assays to test the hypothesis that DNA polymerase errors are a potential source of mutations observed at STR loci in vivo. Bimodal target sequence containing an STR motif and a unique sequence motif will be constructed by inserting dinucleotide and tetranucleotide STR sequences in-frame within the 5 prime coding region of the Herpes simplex virus thymidine kinase (HSV-tk) gene. DNA sequences that make up the ATP-binding site constitute the unique DNA control for which to compare directly the frequency of errors at the STR motif. These targets will be used as DNA templates during in vitro DNA synthesis catalyzed by human DNA polymerase beta and the calf thymus polymerase alpha-primase complex. The mutations produced will be analyzed to quantitate the relative frequencies of polymerase-mediated errors in the two motifs and to ascertain precise in vitro polymerase error rates in STR sequences as a function of repeat size and base composition. The bimodal targets sequences also will be incorporated into ori-rho-tk shuttle vectors which replicate episomally in human lymphoblastoid cells. Mutations produced during replication of the shuttle vectors under defined conditions of leading and lagging strand DNA replication in culture normal cells will be analyzed to determine quantitative mutation rates for STR loci as a function of sequence composition. The bimodal shuttle vectors will be used to demonstrate whether DNA adducts produced by two distinct carcinogens, the arylaminating agent N- benzoyloxy-N-methyl-4- aminoazobenzene and the alkylating agent N- ethyl-N-nitrosourea, can induce mutations in the STR motifs. Two pathways for mutation induction will be followed, the gain/loss of integral repeat units in continuous repeat arrays and base substitution mutations in discontinuous repeat arrays. Data generated by this proposal will: I) enhance our understanding of the mechanisms of human somatic cell mutagenesis by providing quantitation of mutation rates in repetitive DNA, and II) establish the degree to which repetitive DNA is destabilized by chemical carcinogens. Our long-term research objective is to test the hypothesis that mutations in repetitive DNA provide an important source of genotypic variation that drives neoplastic progression.