Label-free electrochemical monitoring of DNA ligase activity.
This study presents a simple, label-free electrochemical technique for the monitoring of DNA ligase activity. DNA ligases are enzymes that catalyze joining of breaks in the backbone of DNA and are of significant scientific interest due to their essential nature in DNA metabolism and their importance to a range of molecular biological methodologies. The electrochemical behavior of DNA at mercury and some amalgam electrodes is strongly influenced by its backbone structure, allowing a perfect discrimination between DNA molecules containing or lacking free ends. This variation in electrochemical behavior has been utilized previously for a sensitive detection of DNA damage involving the sugar-phosphate backbone breakage. Here we show that the same principle can be utilized for monitoring of a reverse process, i.e., the repair of strand breaks by action of the DNA ligases. We demonstrate applications of the electrochemical technique for a distinction between ligatable and unligatable breaks in plasmid DNA using T4 DNA ligase, as well as for studies of the DNA backbone-joining activity in recombinant fragments of E. coli DNA ligase.
Vacek
Jan
J
Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Královopolská 135, 612 65 Brno, Czech Republic.
Cahova
Katerina
K
Palecek
Emil
E
Bullard
Desmond R
DR
Lavesa-Curto
Manuel
M
Bowater
Richard P
RP
Fojta
Miroslav
M
D053904:DNA Breaks, Single-Stranded; D011088:DNA Ligases / Q000032:analysis / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism; D055664:Electrochemical Techniques / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology
D004278:DNA, Superhelical; D011088:DNA Ligases