SEQUENCE - SPECIFIC REPAIR OF ALKYLATED CELLULAR DNA

这项研究的长期目标是研究如何消除 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从活性基因中优先去除DNA加合物 与特定病变阻断RNA合成或 负责清除的修复途径。根据文献记录 that the removal去除of replication复制and transcripton转录blocking阻断cyclobutane环丁烷 来自中国人二氢叶酸还原酶基因座的嘧啶二聚体 仓鼠卵巢B11细胞基本上限于转录链 这个基因。7-甲基鸟嘌呤，一种无害的病变， 核酸合成，不优先从该基因座去除。 本研究的具体目的是：（1）确定是否去除 3-甲基腺嘌呤，一种烷基化的碱基， 体外合成，优先来自活跃转录的基因;（2） 为了测试3-甲基腺嘌呤从转录的 （3）确定是否去除活性位点; 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 被长链烷基破坏，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;和（5）研究烷基磷酸三酯损伤， 抑制核酸合成但总体上不能很好地修复 细胞DNA，评估它们从活跃的遗传基因座中的去除。 数据 在这个项目中获得的将有助于确定的一般性， 优先修复，并帮助阐明机制， 现象 两个重要的技术将用于这些 研究：（1）甲氧胺还原产生的脱嘌呤位点 N-甲基嘌呤去除将用于提供对照，以及（2） 变性脉冲场凝胶电泳将被用来检查 大片段DNA的修复（i. e. >25kb）。 后一种技术 将有助于防止对毒性剂量的烷基化剂的需要， 还增强了优先DNA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.