Studies of Chemically Labile Alkylation Damage in DNA

摘要 烷基化试剂造成的烷基化DNA损伤会促进突变和癌症的发展。鸟嘌呤N7 是广泛的烷基化诱变剂、致癌物和抗癌剂的靶标，产生阳离子 N7-烷基鸟嘌呤(N7-烷基G)加合物为主要病变。这些损伤的半衰期为几小时到几天。 因此可以影响DNA的复制和转录。带正电的N7-烷基G损伤也可以 进行进一步的修饰以产生继发性损害，如烷基-甲酰胺基嘧啶(烷基-FapyG) 加合物。许多诱变剂/致癌物诱导的N7-的识别、修复和突变机制 烷基G和烷基-FapyG病变，但N7-黄曲霉毒素B1-G和黄曲霉毒素B1-FapyG等少数病变除外 加合物的特征仍然很差，因此无法完全理解这些加合物的贡献 主要损害到突变和癌症的发展。例如，优势基因的诱变特性 由致癌环氧苯乙烷产生的N7-烷基G加合物尚不清楚。这种知识鸿沟已经 部分是由于制备特定部位的N7-烷基G-和烷基-FapyG-的技术困难 DNA，这归因于N7-烷基G核苷的快速脱嘌和烷基-G-核苷的异构化。 固相DNA合成过程中的FapyG。为了克服N7-烷基G核苷的稳定性问题，我们有 开发了一种2‘-氟技术，通过增加N7-的稳定性来防止自发脱氢 烷基G核苷。为了解决烷基-FapyG的异构化问题，我们采取了一种后合成 从含有N7-烷基G的DNA中产生含有烷基-FapyG的DNA的方法。我们的初步研究 表明鸟嘌呤N7烷基化可以通过促进稀有的形成而影响碱基配对的性质 烯醇互变异构体、同源碱基构象和/或插层。我们的中心假设是N7-烷基G和烷基- FapyG加合物通过改变FapyG加合物的碱基配对性质促进突变和癌症的发展 鸟嘌呤受损。我们的长期研究目标是阐明化学不稳定性对生物的影响 使用创新方法的烷基化损伤及其继发性损伤，如2‘-F化学， POLβ主-客体-络合物体系，以及合成后的DNA修饰。我们的目标是解剖生物 强烷基化诱变剂和抗癌药物诱发的N7-烷基G和烷基-FapyG损伤的后果 尼古丁特有的亚硝胺、环氧苯乙烷、氮芥末和N-甲基苄基亚硝胺等。 为了实现这一目标，我们将表征碱基配对的性质以及识别、突变、 以及N7-烷基G和烷基-FapyG加合物的修复机理 结构生物学和细胞方法。拟议方案的成功执行将极大地 增进我们对致癌物/药物诱导的N7-烷基G和烷基-FapyG损伤对 碱基对的构象、稳定性、互变异构化、突变、识别和修复，从而提供重要的 对烷基化损伤导致的突变和癌症发展的洞察。

ABSTRACT Alkylation DNA damage caused by alkylating agents promotes mutations and cancer development. Guanine N7 is targeted by a wide range of alkylating mutagens, carcinogens, and anticancer agents, producing the cationic N7-alkylguanine (N7-alkylG) adducts as major lesions. These lesions have half-lives of several hours to days in DNA and thus can affect DNA replication and transcription. The positively charged N7-alkylG lesions can also undergo further modification to generate secondary lesions such as alkyl-formamidopyrimidine (alkyl-FapyG) adducts. The recognition, repair, and mutagenesis mechanisms of many mutagen/carcinogen-induced N7- alkylG and alkyl-FapyG lesions, except for a few lesions such as N7-aflatoxin B1-G and aflatoxin B1-FapyG adducts, remain poorly characterized, thereby precluding a complete understanding of the contribution of these major lesions to mutations and cancer development. For example, the mutagenic properties of the predominant N7-alkylG adducts produced by the cancer-promoting styrene oxide are unknown. This knowledge gap has been due in part to the technical difficulty in preparing a site-specific N7-alkylG- and alkyl-FapyG-containing DNA, which is ascribed to the rapid depurination of N7-alkylG nucleosides and the facile isomerization of alkyl- FapyG during solid-phase DNA synthesis. To overcome the stability issue of N7-alkylG nucleosides, we have developed a 2’-fluorine technology that prevents spontaneous depurination by increasing the stability of N7- alkylG nucleosides. To solve the isomerization problem of alkyl-FapyG, we have taken a post-synthetic approach that produces alkyl-FapyG-containing DNA from N7-alkylG-containing DNA. Our preliminary studies show that guanine N7 alkylation can influence base-pairing properties by facilitating the formation of the rare enol tautomer, syn base conformation, and/or intercalation. Our central hypothesis is that N7-alkylG and alkyl- FapyG adducts promote mutations and cancer development by altering the base-pairing properties of the damaged guanine. Our long-term research goal is to elucidate the biological impacts of chemically labile alkylation damages and their secondary lesions using innovative approaches such as the 2’-F chemistry, the polβ host-guest-complex system, and post-synthetic DNA modification. The objective is to dissect the biological consequences of N7-alkylG and alkyl-FapyG lesions induced by potent alkylating mutagens and anticancer agents such as nicotine-specific nitrosamine, styrene oxide, nitrogen mustards, and N-methylbenzyl nitrosamine. To accomplish this objective, we will characterize the base-pairing properties and the recognition, mutagenesis, and repair mechanisms of N7-alkylG and alkyl-FapyG adducts using combined tools of synthetic, biochemical, structural biology, and cellular approaches. The successful execution of the proposed programs will greatly advance our knowledge of the impact of carcinogen/drug-induced N7-alkylG and alkyl-FapyG lesions on the base pair conformation, stability, tautomerism, mutagenesis, recognition, and repair, thereby providing important insights into the alkylation damage-induced mutations and cancer development.