Action mechanism of Escherichia coli DNA photolyase. I. Formation of the enzyme-substrate complex.
Escherichia coli DNA photolyase (photoreactivating enzyme) is a flavoprotein. The enzyme binds to DNA containing pyrimidine dimers in a light-independent step and, upon illumination with 300-600 nm radiation, catalyzes the photosensitized cleavage of the cyclobutane ring thus restoring the integrity of the DNA. We have studied the binding reaction using the techniques of nitrocellulose filter binding and flash photolysis. The enzyme binds to dimer-containing DNA with an association rate constant k1 estimated by two different methods to be 1.4 X 10(6) to 4.2 X 10(6) M-1 S-1. The dissociation of the enzyme from dimer-containing DNA displays biphasic kinetics; for the rapidly dissociating class of complexes k2 = 2-3 X 10(-2) S-1, while for the more slowly dissociating class k2 = 1.3 X 10(-3) to 6 X 10(-4) S-1. The equilibrium association constant KA, as determined by the nitrocellulose filter binding assay and the flash photolysis assay, was 4.7 X 10(7) to 6 X 10(7) M-1, in reasonable agreement with the values predicted from k1 and k2. From the dependence of the association constant on ionic strength we conclude that the enzyme contacts no more than two phosphodiester bonds upon binding; this strongly suggests that the pyrimidine dimer is the main structural determinant of specific photolyase-DNA interaction and that nonspecific ionic interactions do not contribute significantly to substrate binding.
D003101:Collodion; D004247:DNA / Q000378:metabolism* / Q000528:radiation effects; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D008027:Light; D008190:Lyases / Q000378:metabolism*; D008854:Microscopy, Electron; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010782:Photolysis; D011740:Pyrimidine Dimers / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology
D011740:Pyrimidine Dimers; D005182:Flavin-Adenine Dinucleotide; D012965:Sodium Chloride; D003101:Collodion; D004247:DNA; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase