Chemistry and Biology of Alkyl Phosphotriester Lesions

摘要 人类基因组不断受到各种内源性和外源性遗传毒性因子的攻击， 这可能导致DNA损伤并损害基因组完整性。烷基化物质构成了一种普遍存在的 一类DNA损伤剂。除了形成核碱基加合物之外，烷基化剂还可以攻击一个 的非碳键氧原子的核苷酸间磷酸基团，以产生骨架烷基化 产品，即烷基磷酸三酯（烷基-PTE）损伤。虽然烷基PTE病变是诱导在 相对较高的频率，很少有人知道他们的修复和生物后果。我们假设 未修复的烷基PTE损伤可能导致癌症和其他人类疾病的发展， 通过抑制DNA复制和转录，损害遗传信息的流动， 细胞中的突变。我们还证实了烷基-PTE损伤对DNA复制和转录的影响， 可以通过与DNA中骨架磷酸盐缀合的烷基的大小来调节。此外，委员会认为， 烷化剂是最早和最广泛使用的癌症化疗剂。因此，我们认为， 关于烷基PTE损伤如何干扰DNA复制和转录的系统研究将揭示 关于暴露于烷基化剂如何促进癌症发展的分子见解，并提供了一个 为设计更好的肿瘤化疗药物提供了重要的知识基础。为了验证上述假设， 我们根据三个具体目标提出实验：目标#1。合成寡脱氧核苷酸 具有不同侧翼序列的位点特异性插入的烷基-PTE损伤的寡核苷酸（ODN）;目的#2。到 研究烷基PTE损伤如何干扰细胞中DNA复制的效率和保真度。目标3。到 检查烷基PTE损伤的转录诱变和修复。拟议的研究是建立在 我们在化学合成携带损伤的ODNs和利用穿梭载体方面的专业知识 评估DNA损伤如何干扰细胞中DNA复制和转录的方法。的 拟议的研究结果将提供有关修复的系统和分子水平的理解 以及烷基PTE损伤对人类健康的影响。因此，拟议的研究将导致重要的 评估人类接触烷化剂风险的知识，了解 这些病变在人类疾病的病因学，并为癌症化疗开发更好的策略。

ABSTRACT The human genome is continuously attacked by a variety of endogenous and exogenous genotoxic agents, which can lead to DNA damage and compromise genomic integrity. Alkylating species constitute a ubiquitous class of DNA damaging agents. Aside from forming nucleobase adducts, alkylating agents can also attack one of the non-carbon-bonded oxygen atoms of internucleotide phosphate group to yield backbone alkylation products, i.e. the alkyl phosphotriester (alkyl-PTE) lesions. Although the alkyl-PTE lesions are induced at relatively high frequencies, very little is known about their repair and biological consequences. We hypothesize that unrepaired alkyl-PTE lesions may lead to the development of cancer and other human diseases by compromising the flow of genetic information through inhibiting DNA replication and transcription, and inducing mutations in cells. We also posit that the effects of the alkyl-PTE lesions on DNA replication and transcription may be modulated by the size of the alkyl group conjugated with the backbone phosphate in DNA. Moreover, alkylating agents are among the earliest and most widely prescribed cancer chemotherapeutic agents. Hence, a systematic investigation about how the alkyl-PTE lesions perturb DNA replication and transcription will reveal molecular insights about how exposure to alkylating agents contributes to cancer development, and provide an important knowledge basis for designing better cancer chemotherapeutic agents. To test the above hypothesis, we propose experiments according to three specific aims: Aim #1. To synthesize oligodeoxyribonucleotides (ODNs) harboring site-specifically inserted alkyl-PTE lesions with different flanking sequences; Aim #2. To investigate how the alkyl-PTE lesions perturb the efficiency and fidelity of DNA replication in cells. Aim #3. to examine the transcriptional mutagenesis and repair of alkyl-PTE lesions. The proposed research is built upon our established expertise in the chemical syntheses of lesion-carrying ODNs and in utilizing shuttle vector methods for the assessment about how DNA lesions perturb DNA replication and transcription in cells. The outcome of the proposed studies will provide a systematic and molecule-level understanding about the repair and human health consequences of the alkyl-PTE lesions. Thus, the proposed research will lead to important knowledge for assessing the risk of human exposure toward alkylating agents, for understanding the roles of these lesions in the etiology of human diseases, and for developing better strategies for cancer chemotherapy.