N2 ALKYIG ADDUCTS--MUTATIONS AND POLYMERASE INTERACTIONS

描述：(改编自调查员的摘要)最近的证据 表明鸟嘌呤氮加成物在乙醇或 使DNA与&gt；3碳烷基发生烷基化的致癌物。这是意料之中的 N_2-烷基鸟嘌呤加合物将显著干扰DNA复制，因为 烷基可以改变Watson-Crick碱基配对；然而，在 目前还没有包含以下内容的DNA的数据寻址复制 N-烷基鸟嘌呤，也不知道这些加合物是否或如何修复。这 提案将弥合我们对DNA烷化剂诱导的理解的差距 通过以下方式进行诱变：1)含有单一N-乙基鸟嘌呤的寡聚脱氧核苷酸， N_2-异丙基鸟嘌呤、O_6-乙基鸟嘌呤和O_6-异丙基鸟嘌呤碱 合成的。2)含有这四种鸟嘌呤加合物的寡核苷酸将被 作为模板用于纯化的人体外催化DNA聚合 Dna聚合酶a、q、e和n。聚合酶m是最近发现的一种“旁路”。 人类DNA聚合酶被认为参与了跨越阻断的复制 损伤。这些实验将比较编码和扩展属性 N_2与O_6的烷基鸟嘌呤加合物。3)含有这四种的寡核苷酸 烷基鸟嘌呤加合物将被特定地结合到双链中 双链突变报告穿梭质粒pLS189。突变频率和 由N_2与O_6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复制后确定的质粒 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的致突变性。4)关于 N_2-烷基鸟嘌呤加合物是否通过短补片碱基切除修复 修复将通过孵化NER缺乏的人的提取物来解决 含烷基鸟嘌呤双链寡核苷酸的成纤维细胞和 测定(32P)-dGTP在加合位置的掺入。数据 由该提案提供的数据将衡量以前 未探索的N_2-烷基鸟嘌呤加成物与已知的诱变剂的比较 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.