title: 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.
authors.affiliation: Department of Chemistry, University of Connecticut, Storrs, Connecticut, United States of America.
authors.forename: Savithri
authors.initials: S
authors.lastname: Weerasooriya
authors.affiliation: Department of Chemistry, University of Connecticut, Storrs, Connecticut, United States of America.
authors.forename: Vijay P
authors.initials: VP
authors.lastname: Jasti
authors.affiliation: Department of Chemistry, University of Connecticut, Storrs, Connecticut, United States of America.
authors.forename: Ashis K
authors.initials: AK
authors.lastname: Basu
mesh_terms: D002460:Cell Line; D004249:DNA Damage; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D009713:Nucleotidyltransferases / Q000378:metabolism; D013014:SOS Response, Genetics
chemical_list: C425929:DNA polymerase zeta; D009713:Nucleotidyltransferases; D004259:DNA-Directed DNA Polymerase