SEQUENCE-DEPENDENT INDUCTION OF BASE-PAIR SUBSTITUTIONS AND FRAMESHIFTS BY PROPANODEOXYGUANOSINE DURING IN-VITRO DNA-REPLICATION

Template primers containing propanodeoxyguanosine (PdG) in two differe nt sequence contexts (C-PdG-C and T-PdG-T) were replicated by the klen ow fragment of DNA polymerase I. The presence of PdG in the template s trand reduced the extent of in vitro DNA synthesis 10(3)-10(4)-fold co mpared with unmodified template primers. Partial blockade was observed 1 base 3' to the adduct and opposite the adduct. Purines were prefere ntially incorporated opposite the adduct; the V-max/K-m values for inc orporation of dGMP were similar in both sequence contexts, whereas the V-max/K-m for dAMP incorporation increased 4.7-fold when the base pai r 3' to PdG was changed from C:G to T:A. Oligonucleotides containing 1 - and S-base deletions were major products of replication in both sequ ence contexts. Full-length products were observed with templates conta ining T-PdG-T but not C-PdG-C. The major full-length product resulted from incorporation of dAMP residues opposite PdG. Kinetic analysis rev ealed that the major factor contributing to the selective incorporatio n of dAMP in full-length products seas preferential extension of templ ate primers containing PdG:dA termini rather than preferential incorpo ration of dAMP opposite PdG. The observation of PdG --> T mutations in the T-PdG-T context but not the C-PdG-C context during in vitro DNA r eplication parallels findings of in vivo experiments that base pair su bstitutions are induced by PdG in the former sequence context but not the latter.