UC Riverside

Endogenous and environmental agents can cause DNA damage in cells. A number of alkylating agents introduce modifications into DNA bases that can be cytotoxic and mutagenic to the cell. N2-alkyl-2’-deoxyguanosine (dG) lesions are a primary type of DNA damage products resulting from alkylation that have been identified in the genome. While most of the DNA damage can be removed through different types of DNA repair pathways, any unrepaired DNA lesions may obstruct replication and transcription processes and further trigger genome instability. However, there is limited understanding regarding the impact of N2-alkyl-dG lesions in cellular processes and the functions of translesion synthesis (TLS) polymerases in modulating genome instability.In Chapter 2, we synthesized ODNs harboring N2-alkyldG lesions and incorporated them into plasmid vectors. By performing in vivo replication assay in wild-type and TLS polymerase-deficient E. coli cells, we found that N2-Et-dG is slightly less blocking to DNA replication than the two isomeric N2-nBu-dG lesions, which display very similar replication bypass efficiencies. Additionally, Pol II and, to a lesser degree, Pol IV and Pol V are required for the efficient bypass of the N2-alkyl-dG adducts, and none of these lesions were mutagenic. In Chapter 3, we used two independent approaches (proximity labeling and affinity pull-down) followed by LC-MS/MS analysis to profile Pol κ-interacting proteins at the proteome-wide level. Our result revealed that Pol κ interacts with DDX23, and Pol κ ChIP-Seq analyses indicated that Pol κ is enriched at R-loop structure loci in chromatin. In addition, Pol κ recruits DDX23 to R-loop sites in chromatin and promotes unwinding of R-loop structure. We also observed an augmented accumulation of R-loops in Pol κ- and DDX23-deficient cells. Together, we discovered an interaction between Pol κ and DDX23, and revealed that the interaction facilitates resolution of R-loops in cells. In Chapter 4, we demonstrated N2-alkyl-dG adducts induce R-loop structure accumulation on human chromatin by conducting immunofluorescence microscopy and sp-KAS-seq analysis. R-ChIP-qPCR assay further illustrates R-loop structures formed on lesion insertion sites in lesion-bearing episomal plasmids and cellular transcription assay results showed the lesion-induced R-loops strongly impede transcription. We also observed the accumulation of double-strand breaks (DSBs) after N2-alkyl-dG incorporation in DDX23-deficient cells. Together, we conclude that N2-alkyl-dG adducts trigger R-loop accumulation and further induce genome instability in human chromatin. Together, the research described in this dissertation reveals the adverse biological consequences of N2-alkyl-dG lesions and provides a much better understanding of the functions of TLS polymerases in modulating replicative bypass of N2-alkyl-dG lesions and maintaining genome stability.