Substrate specificity of DNA polymerases. I. Enzyme-catalysed incorporation of 5-'1-alkenyl)-2'-deoxyuridines into DNA.
A series of (E)-5-(1-alkenyl)-dUTPs as well as 5-vinyl-and (Z)-5-(1-propenyl)-dUTP have been synthesized to study steric requirements in DNA polymerase reactions. Experiments were carried out in E. coli DNA polymerase I Klenow fragment enzyme system. Substrates were characterized by KM and Vmax-values, initial incorporation rates as well as by total extent of incorporation of the analogues into poly(dA-dT) as a template-primer. Incorporation of the analogues could be best correlated with Vmax-values as well as the very similar initial incorporation rate values. Reactivity (Vmax/KM) showed no correlation with the extent of incorporation. 5-Vinyl-dUTP proved to be as good a substrate of the enzyme as dTTP, whereas (E)-5-(1-heptenyl)-and (E)-5-(1-octenyl)-dUTPs were very poor substrates, their incorporation was strongly limited and they also proved to be very efficient inhibitors of DNA replication, as shown by Ki-values. Substrate specificity of the Klenow enzyme can be explained by the steric hindrance of C-5 substituent, by the "orientational steric substituent effect" concept.
D000475:Alkenes / Q000138:chemical synthesis / Q000378:metabolism; D001665:Binding Sites; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003856:Deoxyuracil Nucleotides / Q000138:chemical synthesis* / Q000378:metabolism; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D013379:Substrate Specificity
D000475:Alkenes; D003856:Deoxyuracil Nucleotides; D007202:Indicators and Reagents; D004259:DNA-Directed DNA Polymerase