Evidence that deoxyribonucleic acid photolyase from bakers' yeast is a flavoprotein

Abstract

DNA photolyase purified from baker's yeast by affinity chromatography on UV-irradiated DNA noncovalently bound to cellulose and by chromatography on activated thiol-Sepharose 4B yields a single protein band having a molecular weight of 51 000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight, 53 000, determined by gel filtration was in good agreement. Upon denaturation of photolyase by heat or 8 M urea, flavin adenine dinucleotide (oxidized) was isolated from the mixture and identified by thin-layer chromatography and spectral analysis. In contrast to flavoproteins to which flavin adenine dinucleotide (oxidized) is bound which generally exhibit two absorbance maxima between 300 and 500 nm, photolyase has only one at 380 nm. These findings and the similar characteristics of the absorbance and emission spectra of native photolyase with those of flavoproteins in which the chromophore is considered to be the 4a,5-reduced flavin have led us to propose this configuration for the photolyase chromophore. The difference in properties of yeast photolyase compared to the one reported previously supports the idea that there are two photolyases in baker's yeast.

Mutations in the NPH1 Locus of Arabidopsis Disrupt the Perception of Phototropic Stimuli

“…For example, the DNA photolyases catalyze the light-dependent repair of UV lightdimerized DNA through the concerted action of two chromophores (for review, see Sancar, 1994). One of the chromophores is an invariant flavin adenine dinucleotide (FAD) molecule (Iwatsuki et al, 1980); the second can be either a deazaflavin (Eker et al, 1981) or pterin derivative (Johnson et al, 1988). It has been shown in vitro under saturating light conditions that light absorption by either the FAD or the deazaflavinl pterin molecule can result in the excision of pyrimidine dimers (Jorns et al, 1990).…”

DNA Repair Enzymes

“…Photolyase I was reported to be a 51-53-kd monomeric enzyme with absorbance and fluorescence properties similar to those describt:d above for Phrl as well an FAD cofactor that was proposed to be a 4a,5-reduced FAD (54). It is likely that Phrl and photolyase I are the same enzyme and that the FAD chromophore was incorrectly identified in the older study due to lability of the blue neutral FAD radical and pterin chromophores .…”

“…Prior to purification of Phrl from an E. coli strain lacking endogenous photolyase: activity (51), variability in the properties of photoylase obtained from commercial preparations of baker's yeast led to the proposal that S. cerevisiae has two photolyases (53,54). Photolyase I was reported to be a 51-53-kd monomeric enzyme with absorbance and fluorescence properties similar to those describt:d above for Phrl as well an FAD cofactor that was proposed to be a 4a,5-reduced FAD (54).…”

Studies of Organic Protein Cofactors Using Electron Paramagnetic Resonance

“…88 All known photolyases contain stoichiometric amounts of non-covalently bound flavin adenine dinucleotide (FAD) as redox-active cofactor. 78,82,85,87,[89][90][91][92][93][94][95] The enzymatic activity of photolyases depends on the photoexcited state of FAD in the fully reduced form, FADH À , see Fig. 9, whereas the vast majority of other flavoproteins utilize the ground state [96][97][98] or the excited state of the fully oxidized redox state of flavin, FAD ox .…”