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基因毒剂的攻击， 这可能会导致DNA损伤并损害基因组的完整性。烷化物种构成了无处不在的 一类DNA损伤剂。除了形成碱基加合物外，烷基化试剂还可以攻击 核苷酸间磷酸基团的非碳键氧原子发生主链烷基化反应 产物，即烷基磷三酯(烷基-PTE)损伤。尽管烷基-PTE损伤是在 频率相对较高，但对它们的修复和生物学后果知之甚少。我们假设 未修复的烷基-PTE损伤可能通过以下方式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流，并诱导 细胞中的突变。我们还假设烷基-PTE损伤对DNA复制和转录的影响 可能受DNA中与主链磷酸共轭的烷基大小的调节。此外， 烷化剂是最早和最广泛使用的癌症化疗药物之一。因此， 一项关于烷基-PTE损伤如何干扰DNA复制和转录的系统研究将揭示 关于接触烷化剂如何促进癌症发展的分子洞察力，并提供了一种 为设计更好的癌症化疗药物提供重要的知识基础。为了检验上述假设， 我们根据三个具体目标提出实验：目的#1.合成寡聚脱氧核苷酸 (ODN)含有具有不同侧翼序列的定点插入的烷基-PTE损伤；目标#2 研究烷基-PTE损伤如何扰乱细胞中DNA复制的效率和保真度。目标3.至 检测烷基-PTE损伤的转录突变和修复。拟议的研究是建立在 我们在携带病变的ODN的化学合成和利用穿梭载体方面拥有成熟的专业知识 DNA损伤如何扰乱细胞内DNA复制和转录的评估方法。这个 拟议的研究结果将提供关于修复的系统和分子水平的理解 以及烷基-PTE损害对人类健康的影响。因此，拟议的研究将导致重要的 评估人类接触烷基化剂的风险的知识，了解 这些损害在人类疾病的病因学上，并用于开发更好的癌症化疗策略。

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.