UC Riverside

DNA carries the genetic information that is essential for all known forms of life. However, under physiological conditions, nucleic acids are chemically unstable and susceptible to assault from endogenous and external sources. Additionally, there are naturally occurring DNA modifications that serve particular biological purposes. Cells are equipped with a plethora of DNA repair mechanisms and DNA damage response pathways to cope with DNA damages. Nevertheless, unrepaired DNA damage poses threats to normal cell function by impeding replication and transcription while introducing mutations during the processes. Translesion synthesis (TLS) is a major type of DNA damage response that utilizes highly versatile DNA polymerases to help bypass and/or extend beyond the damage site. In this thesis, I employed a shuttle vector- and mass spectrometry-based assay to measure the effects of alkylated DNA damage and a natural DNA modification on the above-mentioned biological processes. In Chapter 2, I found that N2-alkyl-2′-deoxyguanosine (N2-alkyl-dG) strongly blocked transcription and elicited CC → AA tandem mutations in nascent transcripts in HEK293T cells. Moreover, genetic ablation of Pol η diminishes the transcriptional bypass efficiencies of the N2-alkyl-dG lesions, which is exacerbated by co-depletion of Rev1. We also observed that the repair of N2-nBu-dG was not pronouncedly affected by genetic depletion of Pol η or Rev1. In Chapter 3, I studied the transcription and repair of a naturally occurring adenine (A) analog, 2,6-diaminopurine (Z), which is employed by many bacteriophages to replace A in their genetic alphabet. The lack of mutagenic consequence and the presence of strong blockage effect of Z on transcription suggest a role of Z in transcriptional regulation. Besides, Z is subjected to removal by transcription-coupled nucleotide-excision repair (TC-NER), but not global-genome NER in human cells. In Chapter 4, I examined DNA backbone damages, where an alkyl group is appended to the internucleotide phosphate group between two thymine bases to form the alkyl phosphotriester (PTE) adducts. I found that the RP diastereomer of nPr-PTEs strongly blocked transcription, and none of the four alkyl-PTEs induced mutant transcripts. Furthermore, polymerase η promotes transcription across the SP-Me-PTE in human cells. Together, these findings provided important molecular insights into biological consequences of DNA modifications in mammalian cells and revealed protein targets in modulating the repair and transcription of these DNA lesions.