Bacteriophage T4 and human type I DNA ligases relax DNA under joining conditions.
Both bacteriophage T4 and human type I DNA ligases in the presence of a mixture of ATP, AMP and PPi altered the topological properties of a supercoiled substrate by a step-wise reaction eventually leading to a population of fully relaxed, covalently closed products. In the presence of only AMP and PPi DNA products containing nicks with 3'OH/5'P termini accumulated in the presence of bacteriophage T4 DNA ligase, suggesting reversal of the entire joining reaction, but not in the presence of human DNA ligase I. Both DNA ligases became deoxyadenylylated in the presence of dATP, but the joining reaction did not proceed to completion. However, with both enzymes the full relaxing reaction took place in the presence of dAMP alone and in the presence of a mixture of dATP, dAMP and PPi. In no case could the joining reaction be reversed by dAMP and PPi. Related experiments with modified derivatives of deoxyribonucleoside 5'-triphosphates and PPi gave results in accord with these observations. The AMP dependent DNA relaxation catalysed by DNA ligases was insensitive to the presence of exonuclease III. These results indicate that controlled relaxation of the substrate by both DNA ligases occurs as a separate reaction rather than by simple reversal of the joining reaction. These findings support the hypothesis that in vivo the DNA topoisomerising ligases relax their substrate at the replication fork both during and separately from ligation of a pre-existing nick.
Ciarrocchi
G
G
Istituto di Genetica Biochimica ed Evoluzionistica, CNR, Pavia, Italy.
Lestingi
M
M
Wright
G
G
Montecucco
A
A
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D017122:Bacteriophage T4 / Q000201:enzymology*; D002384:Catalysis; D011088:DNA Ligases / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D006801:Humans; D009690:Nucleic Acid Conformation
D004270:DNA, Circular; D004278:DNA, Superhelical; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D011088:DNA Ligases