N2 ALKYIG ADDUCTS--MUTATIONS AND POLYMERASE INTERACTIONS

描述：（改编自研究者的摘要）最近的证据 表明在乙醇存在下形成鸟嘌呤 N2 加合物或 具有 >3 个碳烷基的 DNA 烷基化致癌物。预计 N2-烷基鸟嘌呤加合物会显着干扰 DNA 复制，因为 烷基能够改变沃森-克里克碱基配对；然而，在 目前还没有数据能够解决 DNA 的复制问题，其中 N2-烷基鸟嘌呤，也不知道这些加合物是否或如何被修复。这个 该提案将缩小我们对 DNA 烷化剂诱导的理解的差距 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、O6-乙基鸟嘌呤和O6-异丙基鸟嘌呤碱基将是 合成的。 2) 含有这四种鸟嘌呤加合物的寡核苷酸将是 用作纯化人体外催化 DNA 聚合的模板 DNA 聚合酶 a、q、e 和 n。聚合酶m是最近发现的“旁路” 人类 DNA 聚合酶被认为参与跨阻断复制 病变。这些实验将比较编码和扩展属性 N2 与 O6 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被特定位点并入双 搁浅突变报告穿梭质粒pLS189。突变频率和 由 N2 与 O6 烷基鸟嘌呤加合物在体内诱导的光谱将是 在正常和核苷酸切除中质粒复制后确定 修复（NER）缺陷的人成纤维细胞。 NER 在调节中的作用 由此可以确定这些加合物引起的诱变。 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 修复将通过培养 NER 缺陷的人类提取物来解决 具有含烷基鸟嘌呤的双链寡核苷酸的成纤维细胞和 确定 (32P)-dGTP 在加合位置的掺入。数据 该提案提供的内容将衡量以前的生物影响 与更广为人知的诱变剂相比，未开发的 N2-烷基鸟嘌呤加合物 O6-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.