Replicative Bypass of Abasic Site in Escherichia coli and Human Cells: Similarities and Differences

Abstract

Abasic [apurinic/apyrimidinic (AP)] sites are the most common DNA damages, opposite which dAMP is frequently inserted (‘A-rule’) in Escherichia coli. Nucleotide insertion opposite the AP-site in eukaryotic cells depends on the assay system and the type of cells. Accordingly, a ‘C-rule’, ‘A-rule’, or the lack of specificity has been reported. DNA sequence context also modulates nucleotide insertion opposite AP-site. Herein, we have compared replication of tetrahydrofuran (Z), a stable analog of AP-site, in E. coli and human embryonic kidney 293T cells in two different sequences. The efficiency of translesion synthesis or viability of the AP-site construct in E. coli was less than 1%, but it was 7- to 8-fold higher in the GZGTC sequence than in the GTGZC sequence. The difference in viability increased even more in pol V-deficient strains. Targeted one-base deletions occurred in 63% frequency in the GZG and 68% frequency in GZC sequence, which dropped to 49% and 21%, respectively, upon induction of SOS. The full-length products with SOS primarily involved dAMP insertion opposite the AP-site, which occurred in 49% and 71% frequency, respectively, in the GZG and GZC sequence. dAMP insertion, largely carried out by pol V, was more efficient when the AP-site was a stronger replication block. In contrast to these results in E. coli, viability was 2 to 3 orders of magnitude higher in human cells, and the ‘A-rule’ was more rigidly followed. The AP-site in the GZG and GZC sequences gave 76% and 89%, respectively, Z→T substitutions. In human cells, targeted one-base deletion was undetectable, and dTMP>dCMP were the next preferred nucleotides inserted opposite Z. siRNA knockdown of Rev1 or pol ζ established that both these polymerases are vital for AP-site bypass, as demonstrated by 36–67% reduction in bypass efficiency. However, neither polymerase was indispensable, suggesting roles of additional DNA polymerases in AP-site bypass in human cells.

In vivo toxicity of nitroaromatics: A comprehensive quantitative structure–activity relationship study

“…Studies have described modes of action that are closely related to our findings: molecular bulk properties and chemical reactivity, which indicate that the nitro-group is the primary toxic group of the nitroaromatics [52]. Such adducts of different nitroaromatics have been well documented [53][54][55][56]. One of the known mechanisms of toxicity to model laboratory animals is the formation of hemoglobin adducts.…”

“…One of the known mechanisms of toxicity to model laboratory animals is the formation of hemoglobin adducts. Such adducts of different nitroaromatics have been well documented [53][54][55][56].…”

Roles of translesion synthesis DNA polymerases in the potent mutagenicity of tobacco-specific nitrosamine-derived O2-alkylthymidines in human cells

“…This is an order of magnitude higher than 4.5% and 2.7%, respectively, TLS of O 2 -Me-dT and O 2 -POB-dT in uninduced E. coli, which increase only 2–4-fold upon induction of SOS [22]. Comparable magnitude of increase in TLS efficiency in human cells compared to E. coli has been reported in TLS of the abasic site [35, 36]. It is conceivable that TLS efficiency for most replication-blocking DNA lesions is greater in human cells than in E. coli , presumably due to an abundance of bypass pols in the former.…”

Unlike Catalyzing Error-Free Bypass of 8-OxodGuo, DNA Polymerase λ Is Responsible for a Significant Part of Fapy·dG-Induced G → T Mutations in Human Cells

“…In each case the progeny were analyzed for mutations using oligonucleotide hybridization, followed by DNA sequencing. 25, 26 …”