Synthesis, structure and biological effects of carcinogen/drug-induced bulky, intercalatable N7-alkylguanine lesions

烷基化诱变剂和致癌物对DNA的共价修饰与癌症密切相关 发展。已知有多种烷基化试剂可攻击DNA以产生N7-烷基鸟嘌呤(N7- 烷基甲酰胺基嘧啶(烷基-FapyG)损害。尽管在这方面取得了重大进展， 病变的化学、生化和/或致突变特性的表征，我们的分子水平 了解致癌物/药物诱导的大容量嵌入型N7-烷基G的结构和生物学效应 除了黄曲霉毒素B1-N7G加合物等少数病变外，烷基-FapyG加合物仍然有限。这 知识差距在一定程度上是由于在生产足够数量的DNA方面的技术限制 含有位点特异性掺入的N7-烷基G；尽管双链DNA中的N7-烷基G损伤有半衰期 几个小时到几天，核苷中的损伤在化学上极不稳定，不能迅速经历 自发脱氨，从而排除了使用固相法合成N7- 含有烷基G的DNA。我们之前开发了一种过渡状态不稳定战略来解决 并首次报道了含N7-烷基G的DNA的晶体结构。我们的 拟议研究的中心假设是巨大的、可插入的N7-烷基G和烷基-FapyG病变 影响DNA结构和生物过程，包括DNA复制和突变。我们的长期合作 研究目标是阐明致癌物/药物诱导的N7-烷基G和N7-烷基G的结构和生物学效应。 N3-烷基腺嘌呤病变。目的是阐明N7-烷基G和烷基-FapyG的影响 DNA损伤对DNA结构、复制和突变的影响以及对DNA修复机制的剖析 损伤。为了实现这一目标，我们提出了合成、结构测定和生物化学。 N7-烷基G和烷基-FapyG损伤的评价 甲基苄基亚硝胺、黄樟素、ptaquiloside、吖啶半芥末ICR-191、氮半芥末和 铂基“类烷基化”试剂。作为实现我们长期目标的下一步，我们设计了 三个具体目标：1)评估N7-烷基G和烷基-FapyG损伤对 双链DNA的结构和稳定性；2)阐明病变的诱变机制；3) 描绘损伤的DNA修复机制。我们的期望是， 拟议的研究将促进我们在分子水平上理解可插层的N7-烷基G的影响 和烷基-FapyG加合物对DNA结构、复制和突变的影响及其修复机制 从而为烷基化诱导的突变和致癌的病因学提供了新的见解。 此外，含N7-烷基G的DNA的晶体结构将有助于基于结构的设计和 开发可改变DNA结构和生物过程的新型烷基化试剂。

Covalent modification of DNA by alkylating mutagens and carcinogens is closely associated with cancer development. A wide variety of alkylating agents are known to attack DNA to produce N7-alkylguanine (N7- alkylG) and alkyl formamidopyrimidine (alkyl-FapyG) lesions. Despite the major advances in the characterization of chemical, biochemical, and/or mutagenic properties of the lesions, our molecular-level understanding of structural and biological effects of carcinogen/drug-induced bulky intercalatable N7-alkylG and alkyl-FapyG adducts is still limited, except for a few lesions such as aflatoxin B1-N7G adducts. This knowledge gap had been due in part to a technical limitation in generating sufficient quantities of DNA containing site-specific incorporated N7-alkylG; although N7-alkylG lesions in duplex DNA have half-lives of several hours to days, the lesions in nucleosides are chemically extremely unstable to rapidly undergo spontaneous depurination, thereby precluding the use of the solid-phase method for the synthesis of N7- alkylG-containing DNA. We previously developed a transition-state destabilization strategy to solve the chemical instability problem and reported the first crystal structure of an N7-alkylG-containing DNA. Our central hypothesis of the proposed research is that bulky, intercalatable N7-alkylG and alkyl-FapyG lesions affect DNA structure and biological processes including DNA replication and mutagenesis. Our long-term research goal is to elucidate the structural and biological effects of carcinogen/drug-induced N7-alkylG and N3-alkyladenine lesions. The objectives here are to elucidate the impacts of N7-alkylG and alkyl-FapyG lesions on DNA structure, replication and mutagenesis and to dissect the DNA repair mechanism of the lesions. To accomplish this objective, we propose synthesis, structure determination, and biochemical evaluation of N7-alkylG and alkyl-FapyG lesions that are induced by potent carcinogens/drugs including N- methylbenzyl nitrosamine, safrole, ptaquiloside, acridine half-mustard ICR-191, nitrogen half-mustard, and a platinum-based “alkylating-like” agent. As a next step for achieving our long-term goals, we have designed three Specific Aims that are 1) Evaluating the impact of the N7-alkylG and alkyl-FapyG lesions on the structure and stability of duplex DNA; 2) Elucidating the mutagenesis mechanisms of the lesions; and 3) Delineating the DNA repair mechanism for the lesions. Our expectation is that the accomplishment of the proposed research would advance our molecular-level understanding of the impact of intercalatable N7-alkylG and alkyl-FapyG adducts on DNA structure, replication and mutagenesis and the repair mechanism of the lesions, thereby providing new insights into the etiology of alkylation-induced mutagenesis and carcinogenesis. In addition, crystal structures of N7-alkylG-containing DNA would facilitate a structure-based design and development of novel alkylating agents that can alter DNA structure and biological processes.