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.