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Chemical Syntheses and Cellular Replication Studies of DNA Alkyl Phosphotriester Lesions


Exposure to endogenous and exogenous factors could give rise to DNA damage. DNA alkylation constitutes a major form of DNA damage, and alkyl phosphotriesters (alkyl-PTEs) are particularly important owing to their relatively high frequencies of occurrence and resistance to repair in mammalian cells. However, little is known about how alkyl-PTEs influence fidelity and efficiency of DNA replication in cells and how translesion synthesis (TLS) polymerases modulate the replicative bypass of these lesions.

In Chapter 2. We synthesized and characterized oligodeoxynucleotides (ODNs) containing site-specifically inserted alkyl-PTEs (Me, Et, nPr and nBu) with different alkyl groups and diastereomeric configurations (Sp and Rp), and assessed how these lesions impact DNA replication in E. coli cells. Our results revealed that Sp-alkyl-PTEs could be efficiently bypassed, whereas their Rp counterparts exhibited moderate blockage effects on DNA replication. Additionally, Sp-Me-PTE was found to induce TTGT and TTGC mutations, which necessitated Ada proteins.

In Chapter 3. We prepared Me- or nBu-PTE-containing ODNs in two diastereomeric forms with different flanking dinucleotides sequences (XT and TX, X=A, C, G) and examined how these lesions were recognized by DNA replication machinery in E. coli cells. We found that Sp-XT-Me-PTEs were efficiently bypassed with similar distributions in replication products (with approximately 85-90% AT and 5-10% TG at XT site). where Ada protein is essential for the generation of the mutagenic products. Additionally, Rp-XT-Me-PTEs and both diastereomers of TX-Me-PTEs displayed error-free bypass with significant blockage effects. We also found that Ada binds strongly to Sp-Me-PTE at AT site, but not TA site. Our results suggest that the role of Ada may be beyond the repair of Sp-Me-PTE lesion and transcriptional modulation of genes involved in repairing alkylated DNA lesions.

In Chapter 4, we analyzed the cytotoxic and mutagenic properties of pyridyloxobutyl (POB)-PTEs in E. coli cells. We demonstrated that POB-PTEs did not display strong impediments to DNA replication, and replicative bypass of POB-PTEs does not require TLS polymerases. In Addition, POB-PTEs were not mutagenic.

Together, the research described in this dissertation constitutes the first chemical syntheses of ODNs harboring stereochemically defined alkyl-PTE lesions, and provides a much better understanding about the biological consequences of alkyl-PTE lesions.

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