Abstract
DinB, the E. coli translesion synthesis polymerase, has been shown to bypass several N 2-alkylguanine adducts in vitro, including N 2-furfurylguanine, the structural analog of the DNA adduct formed by the antibacterial agent nitrofurazone. Recently, it was demonstrated that the Fe(II)- and α-ketoglutarate-dependent dioxygenase AlkB, a DNA repair enzyme, can dealkylate in vitro a series of N 2-alkyguanines, including N 2-furfurylguanine. The present study explored, head to head, the in vivo relative contributions of these two DNA maintenance pathways (replicative bypass vs. repair) as they processed a series of structurally varied, biologically relevant N 2-alkylguanine lesions: N 2-furfurylguanine (FF), 2-tetrahydrofuran-2-yl-methylguanine (HF), 2-methylguanine, and 2-ethylguanine. Each lesion was chemically synthesized and incorporated site-specifically into an M13 bacteriophage genome, which was then replicated in E. coli cells deficient or proficient for DinB and AlkB (4 strains in total). Biochemical tools were employed to analyze the relative replication efficiencies of the phage (a measure of the bypass efficiency of each lesion) and the base composition at the lesion site after replication (a measure of the mutagenesis profile of each lesion). The main findings were: 1) Among the lesions studied, the bulky FF and HF lesions proved to be strong replication blocks when introduced site-specifically on a single-stranded vector in DinB deficient cells. This toxic effect disappeared in the strains expressing physiological levels of DinB. 2) AlkB is known to repair N 2-alkylguanine lesions in vitro; however, the presence of AlkB showed no relief from the replication blocks induced by FF and HF in vivo. 3) The mutagenic properties of the entire series of N 2-alkyguanines adducts were investigated in vivo for the first time. None of the adducts were mutagenic under the conditions evaluated, regardless of the DinB or AlkB cellular status. Taken together, the data indicated that the cellular pathway to combat bulky N 2-alkylguanine DNA adducts was DinB-dependent lesion bypass.
Highlights
The genome is vulnerable to damage from exogenous and endogenous chemical reactions, including alkylation, oxidation, and deamination [1,2]
DinB Bypasses FF and HF Lesions In Vivo The competitive replication of adduct bypass (CRAB) assay is a quantitative tool used to determine to what extent a given lesion blocks DNA replication in vivo (Figure 1B) [57]
The CRAB assay was performed on the N2dG lesions (m2G, e2G, FF, and HF) in E. coli strains that capture all possible combinations of DinB and AlkB proficiency/deficiency
Summary
The genome is vulnerable to damage from exogenous and endogenous chemical reactions, including alkylation, oxidation, and deamination [1,2]. Several different lesion tolerance and repair pathways have evolved to deal with these types of DNA damage. DNA adduct bypass by translesion synthesis (TLS) polymerases allows for genome replication in the presence of DNA damage, while canonical DNA repair pathways, which include direct repair, base-excision repair, nucleotideexcision repair, non-homologous end joining and homologous recombination remove such damage prior to replication. The present work explored the in vivo consequences (replication efficiency and fidelity) and genetic requirements (presence or absence of bypass polymerases or DNA repair enzymes) of four N2-guanine DNA alkyl adducts: N2-furfurylguanine (FF), 2tetrahydrofuran-2-yl-methylguanine (HF), 2-methylguanine (m2G), and 2-ethylguanine (e2G) (Figure 1A). The 2-fluoro atom (shown in blue) was substituted with an amine group in parallel reactions with
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