title: Rhodobacter sphaeroides CryB is a bacterial cryptochrome with (6-4) photolyase activity.
abstract: Photolyases are efficient DNA repair enzymes that specifically repair either cyclobutane pyrimidine dimers or (6-4) photoproducts in a light-dependent cleavage reaction. The closely related classical cryptochrome blue light photoreceptors do not repair DNA lesions; instead they are involved in regulatory processes. CryB of Rhodobacter sphaeroides was until now described as a cryptochrome that affects light-dependent and singlet oxygen-dependent gene expression and is unusual in terms of its cofactor composition. Here we present evidence for a repair activity of (6-4) photoproducts by CryB and suggest a dual character combining the functions of cryptochromes and photolyases. We investigated the effects of crucial amino acids involved in cofactor or DNA lesion binding on the light-dependent recovery of cells after UV light exposure (in vivo photoreactivation). Remarkably, impairment of one of the two light absorbing cofactors, FAD or 6,7-dimethyl-8-ribityllumazine, only marginally affected the final survival rate but strongly decelerated photoreactivation kinetics. The impairment of both of them together through mutagenesis decreased CryB-dependent photoreactivation to the level of the ∆cryB knockout strain. The third cofactor, a [4Fe4S] iron-sulfur cluster, is indispensable for the structural integrity of the protein. The reduction of FAD via the conserved tryptophan W338, which is crucial for in vitro reduction and consequently DNA repair, is not required for in vivo photoreactivation, suggesting that this reduction pathway to FAD is dispensable in the cellular environment. This demonstrates that in vitro experiments give only limited information on in vivo photolyase activity.
authors.affiliation: Institute of Microbiology and Molecular Biology, Giessen University, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: von Zadow
authors.affiliation: Structural Biochemistry, Department of Chemistry, Philipps University Marburg, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Ignatz
authors.affiliation: Department of Plant Physiology and Photobiology, Faculty of Biology, Philipps University Marburg, Germany.
authors.forename: Richard
authors.initials: R
authors.lastname: Pokorny
authors.affiliation: Structural Biochemistry, Department of Chemistry, Philipps University Marburg, Germany.
authors.forename: Lars-Oliver
authors.initials: LO
authors.lastname: Essen
authors.affiliation: Institute of Microbiology and Molecular Biology, Giessen University, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Klug
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis / Q000528:radiation effects; D015153:Blotting, Western; D056931:Cryptochromes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D050296:Microbial Viability / Q000187:drug effects / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D000072417:Protein Domains; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014466:Ultraviolet Rays
chemical_list: D001426:Bacterial Proteins; D056931:Cryptochromes; D005182:Flavin-Adenine Dinucleotide; D004255:Deoxyribodipyrimidine Photo-Lyase
keywords: 
  Rhodobacter sphaeroides
  ; DNA repair; cryptochrome; iron-sulfur protein; photolyase