Properties of a DNA-adenylate complex formed in the reaction between mammalian DNA ligase I and DNA containing single-strand breaks.
The major DNA ligase from calf thymus (mammalian DNA ligase I) forms a covalent enzyme-AMP complex on incubation with ATP [Söderhäll & Lindahl, J. Biol. Chem. 248, 672-675, (1973)]. The reaction of this complex with DNA has now been studied. When the ligase-adenylate complex is incubated at 0 degrees C for short time periods with DNA containing single-strand breaks, a DNA-AMP complex can be isolated from the reaction mixture by isopycnic centrifugation in CsCl. Incubation at pH 6.5 increased the amount of DNA-AMP complex that could be isolated 10-20-fold relative to that obtained at pH 7.4. Under the same conditions, incubation of the ligase-AMP complex with DNA free from single-strand breaks did not lead to detectable DNA-AMP formation. The DNA-AMP complex was resistant to treatment with dilute acid and alkali indicating the presence of a covalent linkage. Further, this complex was sensitive to DNase but resistant to pronase and RNase. Free AMP was released on further incubation of the isolated DNA-AMP complex with thymus DNA ligase I and Mg2+, suggesting that the complex is a reaction intermediate. Degradation of the DNA-AMP complex with several reagent enzymes indicated that the AMP residues were bound at the 5' ends of the single-strand breaks in DNA by pyrophosphate bonds.
D000249:Adenosine Monophosphate / Q000378:metabolism*; D000469:Alkaline Phosphatase; D000818:Animals; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D004269:DNA, Bacterial / Q000378:metabolism; D004277:DNA, Single-Stranded; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D010727:Phosphoric Diester Hydrolases; D011118:Polynucleotide Ligases / Q000378:metabolism*; D011402:Pronase; D013950:Thymus Gland / Q000201:enzymology*
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D000249:Adenosine Monophosphate; D004247:DNA; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases; D011402:Pronase; D011118:Polynucleotide Ligases