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Kinetic studies of DNA cleavage reactions catalyzed by an ATP-dependent deoxyribonuclease on a 27-MHz quartz-crystal microbalance.
- Source :
-
Biochemistry [Biochemistry] 2005 Feb 22; Vol. 44 (7), pp. 2262-70. - Publication Year :
- 2005
-
Abstract
- Catalytic DNA cleavage reactions by an ATP-dependent deoxyribonuclease (DNase) from Micrococcus luteus were monitored directly with a DNA-immobilized 27-MHz quartz-crystal microbalance (QCM). The 27-MHz QCM is a very sensitive mass-measuring device in aqueous solution, as the frequency decreases linearly with increasing mass on the electrode at a nanogram level. Three steps in ATP-dependent DNA hydrolysis reactions, including (1) binding of DNase to the end of double-stranded DNA (dsDNA) on the QCM electrode (mass increase), (2) degradation of one strand of dsDNA in the 3' --> 5' direction depending on ATP (mass decrease), and (3) release of the enzyme from the nonhydrolyzed 5'-free-ssDNA (mass decrease), could be monitored stepwise from the time dependencies of QCM frequency changes. Kinetic parameters for each step were obtained as follows. The binding constant (K(a)) of DNase to the dsDNA was determined as (28 +/- 2) x 10(6) M(-)(1) (k(on) = (8.0 +/- 0.3) x 10(3) M (-)(1) s(-)(1) and k(off) = (0.29 +/-0.01) x 10(-)(3) s(-)(1)), and it decreased to (0.79 +/- 0.16) x 10(6) M(-)(1) (k'(on) = (2.3 +/- 0.2) x 10(3) M (-)(1) s(-)(1) and k'(off) = (2.9 +/- 0.1) x 10(-)(3) s(-)(1)) for the completely nonhydrolyzed 5'-free ssDNA. This is the reason the DNase bound to the dsDNA substrate can easily release from the nonhydrolyzed 5'-free-ssDNA after the complete hydrolysis of the 3' --> 5' direction of the complementary ssDNA. K(a) values depended on the DNA structures on the QCM, and the order of these values was as follows: the dsDNA having a 4-base-mismatched base-pair end (3) > the dsDNA having a 5' 15-base overhanging end (2) > the dsDNA having a blunt end (1) > the ssDNA having a 3'-free end (4) >> the ssDNA having a 5'-free end (5). Thus, DNase hardly recognized the free 5' end of ssDNA. Michaelis-Menten parameters (K(m) for ATP and k(cat)) of the hydrolysis process also could be obtained, and the order of k(cat)/K(m) was as follows: the dsDNA having a blunt end (1) approximately the dsDNA having a 4-base-mismatched base-pair end (3) > the ssDNA having a free 3' end (4) >> the ssDNA having a free 5' end (5). Thus, DNase could not recognize and not hydrolyze the free 5' end of ssDNA. The DNA hydrolysis reaction could be driven by dATP and GTP (purine base) as well as ATP, whereas the cleavage efficiency was very low driven with UTP, CTP (pyrimidine base), ADP, and AMP.
- Subjects :
- Adenosine Diphosphate chemistry
Base Sequence
Binding Sites
Catalysis
Cytidine Triphosphate chemistry
DNA, Bacterial metabolism
DNA, Single-Stranded chemistry
DNA, Single-Stranded metabolism
Deoxyadenine Nucleotides chemistry
Enzymes, Immobilized chemistry
Enzymes, Immobilized metabolism
Exodeoxyribonuclease V metabolism
Hydrolysis
Kinetics
Microchemistry methods
Micrococcus luteus
Models, Chemical
Molecular Sequence Data
Quartz
Uridine Triphosphate chemistry
Adenosine Triphosphate chemistry
DNA, Bacterial chemistry
Exodeoxyribonuclease V chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 44
- Issue :
- 7
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 15709738
- Full Text :
- https://doi.org/10.1021/bi048486+