Studies of Chemically Labile Alkylation Damage in DNA

摘要 由烷化剂引起的烷化剂DNA损伤促进突变和癌症发展。鸟嘌呤N7 被广泛的烷基化诱变剂、致癌剂和抗癌剂靶向，产生阳离子 N7-烷基鸟嘌呤（N7-alkylG）加合物为主要病变。这些病变的半衰期为数小时至数天， 因此可以影响DNA的复制和转录。带正电荷的N7-烷基G损伤也可以 进行进一步修饰以产生继发性损伤，如烷基-甲酰胺基嘧啶（烷基-FapyG） 加合物许多诱变剂/致癌剂诱导的N7- 烷基G和烷基-FapyG病变，除了少数病变如N7-黄曲霉毒素B1-G和黄曲霉毒素B1-FapyG 加合物，仍然很差的特点，从而排除了一个完整的了解这些贡献， 突变和癌症发展的主要病变。例如，占主导地位的 由促进癌症的氧化苯乙烯产生的N7-烷基G加合物是未知的。这种知识差距 部分是由于制备位点特异性的含N7-烷基G-和烷基-FapyG-的化合物的技术困难， DNA，这归因于N7-烷基G核苷的快速脱嘌呤和烷基G核苷的容易异构化。 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 开发了一种2 '-氟技术，通过增加N7-的稳定性来防止自发脱嘌呤。 烷基G核苷。为了解决烷基-FapyG的异构化问题，我们采取了后合成方法， 从含N7-烷基G的DNA产生含烷基-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碱基的形成来影响碱基配对特性， 烯醇互变异构体、顺式碱构象和/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的碱基配对特性来促进突变和癌症发展。 损坏的鸟嘌呤。我们的长期研究目标是阐明化学不稳定的生物学影响， 烷基化损伤及其继发性损伤，使用创新的方法，如2 '-F化学， polβ宿主-客体-复合物系统，以及合成后DNA修饰。我们的目标是剖析 由强效烷化剂和抗癌诱变剂诱导的N7-烷基G和烷基-FapyG病变的后果 试剂如氧化苯乙烯和氮芥。为了实现这一目标，我们将描述基地- N7-alkylG和alkyl-FapyG的配对性质以及识别、诱变和修复机制 加合物使用合成，生物化学，结构生物学和细胞方法的组合工具。的 成功执行拟议的计划将大大提高我们对影响的认识， 致癌物/药物诱导的N7-烷基G和烷基-FapyG对碱基对构象，互变异构， 诱变，识别和修复，从而提供了重要的见解烷基化损伤诱导的 突变和癌症发展。

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 styrene oxide and nitrogen mustards. 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, tautomerism, mutagenesis, recognition, and repair, thereby providing important insights into the alkylation damage-induced mutations and cancer development.