Making AppDNA using T4 DNA ligase.
5('),5(')-Adenylyl pyrophosphoryl DNA (AppDNA) contains a high-energy pyrophosphate linkage and can be exploited as an activated DNA substrate to derive new DNA enzymes for carrying out various DNA modification reactions. For this reason, enzymatic synthesis of AppDNA is highly desirable. AppDNA is a known intermediate in DNA ligase mediated DNA ligation reactions, but rarely accumulates under normal reaction conditions. Here we report that T4 DNA ligase can quantitatively convert 5(')-phosphoryl DNA donor into AppDNA in the absence of acceptor DNA but in the presence of a template DNA that contains at least one unpaired nucleotide opposite the 5(')-phosphoryl DNA donor site. This adenylylation behavior of T4 DNA ligase is not observed with Thermus aquaticus (Taq) and Escherichia coli DNA ligases. We further found that a donor-template duplex of 11-bp in length is required by T4 DNA ligase for the formation of AppDNA.
Chiuman
William
W
Department of Biochemistry/Department of Chemistry, Health Sciences Centre, McMaster University, 1200 Main Street West, Hamilton, Ont., Canada L8N 3Z5.
Li
Yingfu
Y
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D020137:Base Pair Mismatch; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000096:biosynthesis* / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics; D011088:DNA Ligases / Q000737:chemistry / Q000378:metabolism*; D010759:Phosphorus Isotopes / Q000378:metabolism; D010766:Phosphorylation; D013698:Templates, Genetic
D010759:Phosphorus Isotopes; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D004247:DNA; D011088:DNA Ligases