Specificity of the nick-closing activity of bacteriophage T4 DNA ligase.
Bacteriophage T4 DNA ligase effectively joins two adjacent, short synthetic oligodeoxyribonucleotides (oligos), as guided by complementary oligo, plasmid and genomic DNA templates. When a single bp mismatch exists at either side of the ligation junction, the efficiency of the enzyme to ligate the two oligos decreases. Mismatch ligation is approximately five-fold greater if the mismatch occurs at the 3' side rather than at the 5' side of the junction. During mismatch ligation the 5' adenylate of the 3' oligo accumulates in the reaction. The level of the adenylate formation correlates closely with the level of the mismatch ligation. Both mismatch ligation and adenylate formation are suppressed at elevated temperatures and in the presence of 200 mM NaCl or 2-5 mM spermidine. The apparent Km for the oligo template in the absence of salt is 0.05 microM, whereas the Km increases to 0.2 microM in the presence of 200 mM of NaCl. In this report, we demonstrate these properties of T4 DNA ligase for oligo pairs complementary to the beta-globin gene at the sequence surrounding the single bp mutation responsible for sickle-cell anemia. Because of the highly specific nature of the nick-closing reaction, ligation of short oligos with DNA ligase can be used to distinguish two DNA templates differing by a single nucleotide.
Wu
D Y
DY
Department of Molecular Biochemistry, Beckman Research Institute of the City of Hope, Duarte, CA 91010.
Wallace
R B
RB
D000249:Adenosine Monophosphate / Q000378:metabolism; D001483:Base Sequence; D011088:DNA Ligases / Q000378:metabolism*; D004260:DNA Repair; D004274:DNA, Recombinant; D005914:Globins / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010766:Phosphorylation; D010957:Plasmids; D011118:Polynucleotide Ligases / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013095:Spermidine / Q000494:pharmacology; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology*; D013698:Templates, Genetic; D013997:Time Factors
D004274:DNA, Recombinant; D009838:Oligodeoxyribonucleotides; D000249:Adenosine Monophosphate; D012965:Sodium Chloride; D005914:Globins; D011088:DNA Ligases; D011118:Polynucleotide Ligases; D013095:Spermidine