Sequence-dependent induction of base pair substitutions and frameshifts by propanodeoxyguanosine during in vitro DNA replication.

Hashim MF, Marnett LJ
J Biol Chem. 1996 271 (15): 9160-5

PMID: 8621568 · DOI:10.1074/jbc.271.15.9160

Template primers containing propanodeoxyguanosine (PdG) in two different sequence contexts (C-PdG-C and T-PdG-T) were replicated by the Klenow fragment of DNA polymerase I. The presence of PdG in the template strand reduced the extent of in vitro DNA synthesis 10(3) - 10(4)-fold compared with unmodified template primers. Partial blockade was observed 1 base 3' to the adduct and opposite the adduct. Purines were preferentially incorporated opposite the adduct; the Vmax/Kmvalues for incorporation of dGMP were similar in both sequence contexts, whereas the Vmax/Km for dAMP incorporation increased 4.7-fold when the base pair 3' to PdG was changed from C:G to T:A. Oligonucleotides containing 1- and 2-base deletions were major products of replication in both sequence contexts. Full-length products were observed with templates containing T-PdG-T but not C-PdG-C. The major full-length product resulted from incorporation of dAMP residues opposite PdG. Kinetic analysis revealed that the major factor contributing to the selective incorporation of dAMP in full-length products was preferential extension of template primers containing PdG:dA termini rather than preferential incorporation 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 replication parallels findings of in vivo experiments that base pair substitutions are induced by PdG in the former sequence context but not the latter.

MeSH Terms (10)

Base Sequence Deoxyguanosine DNA Adducts DNA Damage DNA Replication Kinetics Models, Biological Molecular Sequence Data Mutagenesis Templates, Genetic

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