Sequence-dependent induction of base pair substitutions and frameshifts by propanodeoxyguanosine during in vitro DNA replication.
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.
Hashim
M F
MF
A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Center in Molecular Toxicology and the Vanderbilt Cancer Center, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Marnett
L J
LJ
D001483:Base Sequence; D018736:DNA Adducts; D004249:DNA Damage; D004261:DNA Replication; D003849:Deoxyguanosine / Q000031:analogs & derivatives*; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D016296:Mutagenesis; D013698:Templates, Genetic
D018736:DNA Adducts; C060934:1,N(2)-propanodeoxyguanosine; D003849:Deoxyguanosine