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.
Highlights
Abasic sites, called apurinic or apyrimidinic sites (AP-sites), are one of the most common lesions in DNA [1]
Analysis of bypass efficiency in E. coli indicated that transelsion synthesis (TLS) of APsite is a rare event and that pol V is required for 60– 70% of the bypass in the GZC sequence, but it plays a less critical role in the GZG sequence
In vitro studies have shown that pol V can insert dAMP opposite an AP-site much more proficiently than pol III or pol IV [22], which was indicated in our in vivo experiments
Summary
Called apurinic or apyrimidinic sites (AP-sites), are one of the most common lesions in DNA [1]. They occur both spontaneously and following chemical modification of DNA [2,3,4,5,6]. AP-sites are non-coding and they strongly block DNA replication by replicative DNA polymerases (pols). A specialized class of pols, known as the transelsion synthesis (TLS) enzymes, can bypass DNA damages, including AP-sites [7]. When a nucleotide is incorporated opposite AP-site, it frequently results in a mutation [8,9]
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