Modified nucleoside 5'-triphosphates containing 2',3'-fused three-membered rings as substrates for different DNA polymerases.
5'-Triphosphates of 1-(2',3'-epithio-2',3'-dideoxy-beta-D- lyxofuranosyl)thymine, 1-(2',3'-epithio-2',3'-dideoxy-beta-D-ribofuranosyl)thymine and 2',3'-lyxoanhydrothymidine have been shown to be termination substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) reverse transcriptases as well as DNA polymerase I from E. coli and DNA polymerase beta from rat liver. At the same time they do not terminate DNA synthesis catalysed by DNA polymerase epsilon from human placenta. Km values of ltTTP, rtTTP and laTTP incorporation into the DNA chain during catalysis by AMV reverse transcriptase agree closely with each other being 1.5-2.5 times higher than Km value for dTTP. Furthermore, Vmax values for modified substrates are only 2-3 times lower than Vmax for dTTP. The evidence favours the hypothesis of high affinity of modified nucleotides with a flattened furanosyl ring for DNA polymerase active sites.
Semizarov
D G
DG
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.
Victorova
L S
LS
Krayevsky
A A
AA
Kukhanova
M K
MK
D000818:Animals; D009189:Avian Myeloblastosis Virus / Q000201:enzymology / Q000235:genetics; D004277:DNA, Single-Stranded; D004259:DNA-Directed DNA Polymerase / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D006678:HIV / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008099:Liver / Q000201:enzymology; D010920:Placenta / Q000201:enzymology; D012194:RNA-Directed DNA Polymerase / Q000032:analysis; D051381:Rats; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000737:chemistry*
D004277:DNA, Single-Stranded; D013942:Thymine Nucleotides; C082289:2',3'-lyxoanhydrothymidine 5'-triphosphate; C082287:1-(2',3'-epithio-2',3'-dideoxy-beta-D-lyxofuranosyl)thymine 5'-triphosphate; C082288:1-(2',3'-epithio-2',3'-dideoxy-beta-D-ribofuranosyl)thymine 5'-triphosphate; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase