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.

Photolyases and Cryptochromes in UV‐resistant Bacteria from High‐altitude Andean Lakes

“…(B) Level of amino acid conservation for same alignment, using a Logo image with the distribution Kullback-Leibler, where the triad of tryptophan showed its conserved nature. (73); this kind of proteins had initially been thought to be absent in prokaryotes and in most eukaryotic organisms (75,76). Photolyase-like genes with FeS clusters were confirmed in the two strains of HAAL analyzed in this article, suggesting a possible photolyase activity for them.…”

“…Then, oxidative damage of the cluster could trigger disordering or structural change of the C‐terminal end and alter the DNA binding, which in turn can act as a photoreactive agent, in a similar way to that proposed to the W‐triad . This hypothesis may be sustained as FeS‐BCP from Rhodobacter sphaeroides showed [6‐4] photolyase activity ; this kind of proteins had initially been thought to be absent in prokaryotes and in most eukaryotic organisms . Photolyase‐like genes with FeS clusters were confirmed in the two strains of HAAL analyzed in this article, suggesting a possible [6‐4] photolyase activity for them.…”

“… and Zadow et al . also described photolyase (6‐4) activity for a FeS‐BCP sequence from A. tumefaciens and Rhodobacter sphaeroides .…”

Redox-Based Transcriptional Regulation in Prokaryotes: Revisiting Model Mechanisms

“…Cryptochromes and photolyases bear a conserved N-terminal a/b domain and an a-helical domain that noncovalently binds an FAD in their catalytic center. The catalytic activity of photolyases requires the FAD to be in its two-electron reduced active state as FADH - (386). The N-terminus might bind other antenna chromophores.…”

“…Also, V. cholerae cryptochrome, VcCry1, has been described as a DASH cryptochome (326). RsCryB exhibits repair activity of photoproducts (386), suggesting a dual character combining the functions of cryptochromes and photolyases. Moreover, RsCryB is a close homologue of the photolyase PhrB from A. tumefaciens.…”