tailieunhanh - Báo cáo khoa học: Kinetics and thermodynamics of nick sealing by T4 DNA ligase

T4DNA ligase is an Mg 2+ -dependent andATP-dependent enzyme that seals DNA nicks in three steps: it covalently binds AMP,transadenylates the nick phosphate,and cata-lyses formation of the phosphodiester bond releasing AMP. In this kinetic study,we further detail the reaction mechan-ism,showing that the overall ligation reaction is a super-impositionof twoparallel processes: a processive ligation,in which the enzyme transadenylates and seals the nickwithout dissociating fromdsDNA,and a nonprocessive ligation,in which the enzyme takes part in the abortive adenylation cycle (covalent binding of AMP,transadenylation of the nick,and dissociation) | Eur. J. Biochem. 270 4315-4325 2003 FEBS 2003 doi Kinetics and thermodynamics of nick sealing by T4 DNA ligase Alexey V. Cherepanov and Simon de Vries Kluyver Department of Biotechnology Delft University of Technology the Netherlands T4 DNA ligase is an Mg2 -dependent and ATP-dependent enzyme that seals DNA nicks in three steps it covalently binds AMP rransadenylates the nick phosphate add ctUa-lyses formation of the phosphodiester bond releasing AMP. In this kinetic study we further detail the reaction mechanism showing tìiat the oe-miH liaudon recciíon is a sLiprr-imposition of two parallel processes a processive ligation in which the enzyme transadenylates and seals the nick without dissociating from dsDNA and a nonproctssive ligation m which the enzyme takes part in the abortive adenylation cycle covalent binding of AMP araasnhanylatian of die nick mid dissociatian . At low cactnnacidans al ATP 10 M and when the DNA nick is sealed with mismatching base pairs . five adjacent tins superimposition resolves into two kinetic phases a burst ligation w min-1 and a subsequent slow ligation w 2 X 10-3 min-1 . The relative rate and extent of each phase depend on the concentrations of ATP and Mg2 . The activation energies of self-adenylation kcal-mol-1 transadenylation of the nick kcal-mol-1 mid nicksealing kcal-mol-1 were determined for several DNA substrates. The low activation energy of transadenylation implies that the transfer of AMP to the terminal DNA phosphate is a spontaneous reaction and that the T4 DNA ligase-AMP complex is a high-energy intermediate. To summarize current findings in the DNA ligation field we delineate a kinetic mechanism of T4 DNA ligase catalysis. Keywords DNA ligase end-joining kinetics mechanism of action mismatching nick. T4 DNA ligase is an enzyme that catalyses formation of the phosphodiester bond between the adjacent 5 -PO. and 3 -OH groups of two dsDNA fragments 1 . It is able

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