Examining the Role of DinBG32 on the DinB Ternary Complex

Abstract

Translesion synthesis (TLS) is a highly conserved DNA damage tolerance pathway that allows for bypass of bulky lesions. Lesions are generated via a variety of environmental and metabolic processes, which result in the stalling of DNA replication and can lead to cell death. TLS Y-family polymerases possess wider active sites than typical replicative polymerases, permitting the insertion of nucleotides opposite of the lesion; however, Y-family polymerases have less stringent geometric base pair checking, misincorporating nucleotides more frequently and thereby operating with lower fidelity. Mutations generated by such misincorporations may give giving rise to antibiotic resistance in bacteria and cancerous activity in eukaryotes. Bacterial DinB, a Y-family polymerase found across all domains of life, is responsible for replication opposite N2-deoxyguanosine (N2-dG) lesions. The replicative activity of DinB, while important, has been known to cause -1 frameshift of DNA. Recently, it has been found that DinB forms a ternary complex with RecA and UmuD2, which increases stability and decreases deleterious frameshift activity. The understanding of the overall structure and function of the ternary complex is still in early stages. In order to further assess current theoretical models of the ternary complex and its role in TLS, we analyzed a significant, highly conserved DinB amino acid residue, DinBG32, on the junction of DinB and RecA. We altered this residue by site-directed mutagenesis to examine the effects of the mutated residue on the behavior of the ternary complex (in comparison to the wild-type complex) in different phenotypic backgrounds and environmental conditions.