SEQUENCE--SPECIFIC REPAIR OF ALKYLATED CELLULAR DNA

这项研究的长期目标是检查移除 来自基因组特定区域的烷基化损伤。这将是 这样做是为了辨别是否优先移除活性基因中的DNA加合物 与特定病变阻断RNA合成的能力或与 负责其清除的修复路径。它已经被记录在案 去除复制和转录子阻断环丁烷 中国人二氢叶酸还原酶基因座上的嘧啶二聚体 仓鼠卵巢B11细胞主要局限于转录的链 这种基因。7-甲基鸟嘌呤，一种无害的病变，不会阻断 核酸合成，并不优先从该基因座移除。 研究的具体目的是：(1)确定移除 由3-甲基腺嘌呤组成，这是一种已知能阻断核酸的烷基化碱基 体外合成，优先于转录活跃的基因；(2) 检测转录产物中3-甲基腺嘌呤的优先去除性 活性位点链；(3)确定是否移除 来自表达基因的3-甲基腺嘌呤依赖于核苷酸切除 或碱基切除修复；(4)确定嘌呤修复是否 被更长的链烷基破坏，这一过程似乎是 依赖于完整的核苷酸切除修复，优先发生在 转录基因；以及(5)研究烷基磷酸三酯损伤，这是 抑制核酸合成，但没有得到很好的修复 细胞DNA，评估它们从活跃的遗传位点上的移除。数据 在本项目中获得的信息将有助于确定 优先修复并有助于阐明其调控机制 现象。两种重要的技术将用在这些 调查：(1)甲氧胺还原生成的无嘌呤中心 来自N-甲基嘌呤的去除将用于供应控制，以及(2) 变性脉冲场凝胶电泳法将用于检测 修复大片段的DNA(即25kb)。后一种技术 将有助于防止需要有毒剂量的烷化剂和 也增强了DNA优先修复方法的通用性。

The long term objectives of the research are to examine the removal of alkylated lesions from specific regions of the genome. This will be done to discern if preferential removal of DNA adducts from active genes is linked to a particular lesion's ability to block RNA synthesis or to the repair pathway responsible for its clearance. It has been documented that the removal of replication and transcripton blocking cyclobutane pyrimidine dimers from the dihydrofolate reductase locus of Chinese hamster ovary B11 cells is limited essentially to the transcribed strand of that gene. 7-Methylguanine, an innocuous lesion that does not block nucleic acid synthesis, is not removed preferentially from this locus. The specific aims of the research are: (1) to determine if the removal of 3-methyladenine, an alkylated base known to block nucleic acid synthesis in vitro, is preferential from actively transcribed genes; (2) to test for preferential removal of 3-methyladenine from the transcribed strand of active loci; (3) to determine if the removal of 3-methyladenine from expressed genes is dependent on nucleotideexcision or base-excision repair; (4) to determine if the repair of purines damaged with longer chain alkyl groups, a process that appears to be dependent on intact nucleotide-excision repair, occurs preferentially in transcribed genes; and (5) to study alkylphosphotriester lesions, which inhibit nucleic acid synthesis but are not well repaired in total cellular DNA, assessing their removal from active genetic loci. Data obtained in this project will aid in determining the generality of preferental repair and help to elucidate the mechanism governing the phenomenon. Two important techniques will be used in these investigations: (1) methoxyamine reduction of apurinic sites generated from N-methyepurine removal will be used to supply controls, and (2) denaturing pulsed-field gel electrophoresis will be employed to examine repair in large segments of DNA (i. e. >25kb). The latter technique will help to prevent the need for toxic doses of alkylating agents and also enhance the versatility of preferential DNA repair methods.