Binding of Escherichia coli DNA photolyase to UV-irradiated DNA

Abstract

Escherichia coli DNA photolyase is a flavoprotein which catalyzes the photomonomerization of pyrimidine dimers produced in DNA by UV irradiation. In vivo, the enzymes acts by a 2-step mechanism: it binds to dimer-containing DNA in a light-dependent reaction and upon exposure to 300-500-nm light breaks the cyclobutane ring and dissociates from the substrate. Using photolyase purified to homogeneity, the 1st step of the reaction, DNA binding was studied in vitro; enzyme-DNA complex formation was quantitated by the nitrocellulose filter binding assay. The enzyme binds specifically to UV-irradiated DNA regardless of whether the DNA is in the superhelical, open circular or linear form or whether the DNA is single or double stranded. The binding reaction is optimum at a NaCl concentration of 125 mM and at pH 7.5. Although, photolyase is retained by the nitrocellulose filters with .apprx. 100% efficiency, the binding efficiency of a single enzyme-substrate complex is .apprx. 0.34. The complexes can be dissociated by exposing them to photoreactivating light either in solution or on the filter.