Sealing of gaps in duplex DNA by T4 DNA ligase.
Single-strand gaps in DNA molecules were found to be a substrate for T4 DNA ligase. Sealing of the gaps was optimal at the same conditions as ligation of blunt-ended DNA molecules. Spermidine at a concentration of 2 mM stimulated the ligation of gaps, as well as the joining of DNA molecules with cohesive and blunt ends. In addition, spermidine reduced the optimal ATP concentration. The ligation of single-stranded gaps was a slow process, reaching a plateau after several hours at 25 degrees C. Approximately 10% of circular duplex plasmid pBR322 DNA molecules with a gap of 1-5 nucleotides could be converted to a covalently closed form. When such molecules were used for transformation of E. coli cells deletion mutants were obtained at a high frequency. The size and position of the gaps and the deletions were equivalent, confirming that T4 DNA ligase was sealing the gaps.
D011088:DNA Ligases / Q000378:metabolism*; D004277:DNA, Single-Stranded; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D009154:Mutation; D010957:Plasmids; D011118:Polynucleotide Ligases / Q000378:metabolism*; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology*
D004277:DNA, Single-Stranded; D011088:DNA Ligases; D011118:Polynucleotide Ligases