Photoenzymatic repair of ultraviolet damage in DNA. II. Formation of an enzyme-substrate complex.

The photoenzyme from bakers' yeast which repairs ultraviolet-inactivated transforming DNA is mechanically bound to ultraviolet-irradiated DNA in the dark, but not to unirradiated DNA. In the bound condition it is stabilized against inactivation by heat and heavy metals. Both the mechanical binding and stabilization are eliminated by illumination. These observations are consistent with the reaction scheme suggested by kinetic studies, in which the enzyme combines with the ultraviolet lesions in DNA and the complex absorbs light, producing repair and subsequent liberation of the enzyme. The approximately exponential decrease of heat stabilization during illumination gives the first order rate constant for the light-dependent step at the corresponding light intensity. This quantity in turn sets limits on the possible magnitude of the molar absorption coefficient of the enzyme-substrate complex and on the quantum yield of the process.