Characterization of an ATP-dependent type I DNA ligase from Arabidopsis thaliana.
Here we report the purification and biochemical characterization of recombinant Arabidopsis thaliana DNA ligase I. We show that this ligase requires ATP as a source for adenylation. The calculated Km [ATP] for ligation is 3 microM. This enzyme is able to ligate nicks in oligo(dT)/poly(dA) and oligo(rA)/poly(dT) substrates, but not in oligo(dT)/poly(rA) substrates. Double-stranded DNAs with cohesive or blunt ends are also good substrates for the ligase. These biochemical features of the purified enzyme show the characteristics typical of a type I DNA ligase. Furthermore, this DNA ligase is able to perform the reverse reaction (relaxation of supercoiled DNA) in an AMP-dependent and PPi-stimulated manner.
Wu
Y Q
YQ
Friedrich Miescher-Institiute, Basel, Switzerland.
Hohn
B
B
Ziemienowic
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology*; D001483:Base Sequence; D011088:DNA Ligases / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D011088:DNA Ligases