N2 ALKYIG ADDUCTS--MUTATIONS AND POLYMERASE INTERACTIONS

描述：（改编自研究者摘要）近期证据 表明鸟嘌呤N2加合物在乙醇存在下形成，或 致癌物质，使具有>3个碳烷基的DNA烷基化。预计 N2-烷基鸟嘌呤加合物会显著干扰DNA复制， 烷基处于改变Watson-Crick碱基配对位置;然而， 目前，没有数据解决DNA的复制， N2-烷基鸟嘌呤，也不知道这些加合物是否或如何修复。这 这些建议将缩小我们对DNA烷化剂诱导 通过以下诱变：1）含有单个N2-乙基鸟嘌呤的寡脱氧核糖核苷酸， N2-异丙基鸟嘌呤、O 6-乙基鸟嘌呤和O 6-异丙基鸟嘌呤碱基将是 合成了2)含有这四种鸟嘌呤加合物的寡核苷酸将是 作为模板，用于纯化的人DNA体外催化聚合 DNA聚合酶a、q、e和n。聚合酶m是最近发现的“旁路” 一种被认为参与复制的人类DNA聚合酶 病变这些实验将比较 N2与O 6烷基鸟嘌呤加合物。3)含有这四种的寡核苷酸 烷基鸟嘌呤加合物将位点特异性地掺入双链中。 链突变报告穿梭质粒pLS 189。突变频率和 由N2与O 6烷基鸟嘌呤加合物在体内诱导的光谱将是 在正常质粒复制和核苷酸切除后测定 修复（NER）缺陷的人成纤维细胞。NER在调节 由此可以确定这些加合物的诱变。4)问题 N2-烷基鸟嘌呤加合物是否通过短补丁碱基切除修复 修复将通过孵育NER缺陷的人的提取物来解决。 用含烷基鸟嘌呤的双链寡核苷酸与成纤维细胞接触， 测定内收位置处（32 P）-dGTP的掺入。数据 该提案提供的数据将衡量以前的生物影响 与已知的诱变剂相比，未探索的N2-烷基鸟嘌呤加合物 O 6-烷基鸟嘌呤加合物。

DESCRIPTION: (Adapted from the investigator's abstract) Recent evidence indicates that guanine N2 adducts form in the presence of ethanol or carcinogens that alkylate DNA with >3 carbon alkyl groups. It is anticipated that N2-alkylguanine adducts will significantly perturb DNA replication because the alkyl group is in position to alter Watson-Crick base pairing; however, at present there are no data addressing replication of DNA containing N2-alkylguanine, nor is it known if or how these adducts are repaired. This proposal will close that gap in our understanding of DNA alkylator-induced mutagenesis by: 1) Oligodeoxyribonucleotides containing single N2-ethylguanine, N2-isopropylguanine, O6-ethyl guanine, and O6-isopropylguanine bases will be synthesized. 2) Oligonucleotides containing these four guanine adducts will be used as templates for DNA polymerization catalyzed in vitro by purified human DNA polymerase a, q, e, and n. Polymerase m is a recently discovered "bypass" human DNA polymerase thought to be involved in replication across blocking lesions. These experiments will compare the coding and extension properties of the N2 vs. O6 alkylguanine adducts. 3) Oligonucleotides containing these four alkylguanine adducts will be incorporated site specifically into the double stranded mutation reporting shuttle plasmid pLS189. The mutation frequency and spectra induced in vivo by the N2 vs. O6 alkylguanine adducts will be determined after replication of the plasmid in normal and nucleotide excision repair (NER)-deficient human fibroblasts. The role of NER in modulating mutagenesis by these adducts can thereby be determined. 4) The question of whether the N2-alkylguanine adducts are repaired by short patch base excision repair will be addressed by incubating extracts of NER-deficient human fibroblasts with alkylguanine-containing double-stranded oligonucleotides and determining incorporation of (32P)-dGTP at the adducted position. The data provided by this proposal will gauge the biologic impact of previously unexplored N2-alkylguanine adducts in comparison to the better known mutagenic O6-alkylguanine adducts.