MUTATIONAL MECHANISMS OF REPETITIVE DNA IN HUMAN CELLS

这项研究提案的目的是评估 短串联重复序列的自发突变和致癌基因突变 (STR)人类细胞的DNA基因座。我们建议利用互补性 体外/体外诱变试验检验DNA 聚合酶错误是STR观察到的突变的潜在来源 活体内的基因座。含有STR基序和 唯一的序列基序将通过插入二核苷酸和 四核苷酸STR序列框内5个素数编码 单纯疱疹病毒胸苷激酶(HSV-tk)基因的区域。 组成ATP结合位点的DNA序列构成了 独一无二的DNA对照，可直接比较 STR主题中的错误。这些靶标将被用作DNA模板 人DNA聚合酶催化的DNA体外合成 β和小牛胸腺聚合酶α-底物酶复合体。这个 将对产生的突变进行分析，以量化相关的 聚合酶介导的错误在两个基序和TO中的频率 确定STR序列中准确的体外聚合酶错误率为 重复大小和碱基组成的函数。双峰目标 序列还将被整合到Ori-Rho-tk穿梭载体中，该载体 在人类淋巴母细胞中进行异体复制。突变 在以下定义的穿梭载体的复制期间产生 细菌培养中链DNA复制的超前和滞后条件 将对正常细胞进行分析，以确定定量突变率 作为序列组成的函数的STR基因座。双峰模式 穿梭载体将被用来证明DNA加合物 由两种不同的致癌物质产生，芳胺化试剂N- 苯甲酰氧基-N-甲基-4-氨基偶氮苯和烷化剂N- 乙基-N-亚硝脲可诱导STR基序发生突变。二 将遵循突变诱导的途径，获得/丢失 连续重复阵列中的整体重复单元和碱基替换 不连续重复序列中的突变。由此生成的数据 建议将：i)加强我们对以下机制的了解 提供突变定量的人体细胞诱变 重复DNA中的比率，以及ii)确定重复的程度 化学致癌物会破坏DNA的稳定性。我们的长期研究 目的是检验重复DNA中突变的假设 提供了一个重要的基因变异来源，推动 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.