Initiator role of double stranded DNA in terminal transferase catalyzed polymerization reactions.
Binding of the 58 kDa monomer and 44 kDa alpha beta dimer forms of terminal deoxynucleotidyl transferase to double stranded DNA was demonstrated by gel retardation and tryptophan fluorescence quenching. The dissociation constants and cooperativity parameters were similar to those that have been determined for binding of these two forms of terminal transferase to single stranded DNA. However, the double stranded DNA binding site size of 10 nucleotides was half the size expected. The efficacy of blunt ended DNA as an initiator in the polymerization reaction catalyzed by terminal transferase was demonstrated by radiometric assays and product analyses on agarose gels. The initial reaction kinetics indicated that dGTP but not dATP was added efficiently to a blunt double stranded DNA 3' end. These results are correlated with current models for in vivo terminal transferase function.
Robbins
D J
DJ
Department of Biochemistry, University of Kentucky Medical Center, Lexington 40536-0084.
Coleman
M S
MS
D001483:Base Sequence; D004247:DNA / Q000378:metabolism* / Q000502:physiology; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism* / Q000502:physiology; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000502:physiology; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D007700:Kinetics; D010442:Peptide Chain Initiation, Translational; D011506:Proteins / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D004277:DNA, Single-Stranded; D011506:Proteins; D004247:DNA; D004254:DNA Nucleotidyltransferases; D004253:DNA Nucleotidylexotransferase; D004259:DNA-Directed DNA Polymerase