Analysis of the formation of AMP-DNA intermediate and the successive reaction by human DNA ligases I and II.
DNA ligation catalyzed by all DNA ligases involves two intermediary steps, the formation of the ligase-AMP and the AMP-DNA complexes. A method was developed to purify and analyze the AMP-DNA intermediate from the DNA ligation reaction catalyzed by DNA ligases. This AMP-DNA complex was maximally accumulated by preincubation of human DNA ligase I or II with ATP, followed by interaction with the DNA substrate for 5 s at 0 degrees C. The gel-purified AMP-DNA complex maintained its property as a ligation intermediate. The AMP was directly linked to the 5'-phosphate of DNA with a pyrophosphate bond. The successive ligation reaction following the AMP-DNA complex formation required DNA ligase and Mg2+ ion but was inhibited by ATP and pyridoxal 5'-phosphate, indicating that the availability of the AMP binding site in the enzyme is essential for the completion of the reaction. Furthermore, the formation of the AMP-DNA complex and the subsequent DNA ligation were substrate specific for human DNA ligases I and II. These data, together with previously reported results, suggest that a major difference between human DNA ligases I and II is in their DNA-binding domains. The methods make it convenient to study in depth the kinetics of the overall DNA ligation.
Yang
S W
SW
Department of Molecular Pathology, University of Texas, M. D. Anderson Cancer Center, Houston 77030.
Chan
J Y
JY
D000249:Adenosine Monophosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D002413:Cations, Divalent; D004247:DNA / Q000378:metabolism*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D054306:Dideoxynucleotides; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism
D002413:Cations, Divalent; D003848:Deoxyguanine Nucleotides; D054306:Dideoxynucleotides; C000607892:LIG1 protein, human; D009243:NAD; D000249:Adenosine Monophosphate; C031709:2',3'-dideoxyguanosine 5'-triphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D011088:DNA Ligases; D000072481:DNA Ligase ATP; D008274:Magnesium