Visualizing the DNA repair process by a photolyase at atomic resolution.

Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.

Structural and Functional Analysis of a Prokaryotic (6-4) Photolyase from the Aquatic Pathogen Vibrio Cholerae†.

Photolyases are flavoproteins, which are able to repair UV-induced DNA lesions in a light-dependent manner. According to their substrate, they can be distinguished as CPD- and (6-4) photolyases. While CPD-photolyases repair the predominantly occurring cyclobutane pyrimidine dimer lesion, (6-4) photolyases catalyze the repair of the less prominent (6-4) photoproduct. The subgroup of prokaryotic (6-4) photolyases/FeS-BCP is one of the most ancient types of flavoproteins in the ubiquitously occurring photolyase & cryptochrome superfamily (PCSf). In contrast to canonical photolyases, prokaryotic (6-4) photolyases possess a few particular characteristics, including a lumazine derivative as antenna chromophore besides the catalytically essential flavin adenine dinucleotide as well as an elongated linker region between the N-terminal α/ß-domain and the C-terminal all-α-helical domain. Furthermore, they can harbor an additional short subdomain, located at the C-terminus, with a binding site for a [4Fe-4S] cluster. So far, two crystal structures of prokaryotic (6-4) photolyases have been reported. Within this study, we present the high-resolution structure of the prokaryotic (6-4) photolyase from Vibrio cholerae and its spectroscopic characterization in terms of in vitro photoreduction and DNA-repair activity.

Serial crystallography captures dynamic control of sequential electron and proton transfer events in a flavoenzyme.

Flavin coenzymes are universally found in biological redox reactions. DNA photolyases, with their flavin chromophore (FAD), utilize blue light for DNA repair and photoreduction. The latter process involves two single-electron transfers to FAD with an intermittent protonation step to prime the enzyme active for DNA repair. Here we use time-resolved serial femtosecond X-ray crystallography to describe how light-driven electron transfers trigger subsequent nanosecond-to-microsecond entanglement between FAD and its Asn/Arg-Asp redox sensor triad. We found that this key feature within the photolyase-cryptochrome family regulates FAD re-hybridization and protonation. After first electron transfer, the FAD•- isoalloxazine ring twists strongly when the arginine closes in to stabilize the negative charge. Subsequent breakage of the arginine-aspartate salt bridge allows proton transfer from arginine to FAD•-. Our molecular videos demonstrate how the protein environment of redox cofactors organizes multiple electron/proton transfer events in an ordered fashion, which could be applicable to other redox systems such as photosynthesis.

Ultrafast photoreduction dynamics of a new class of CPD photolyases.

NewPHL is a recently discovered subgroup of ancestral DNA photolyases. Its domain architecture displays pronounced differences from that of canonical photolyases, in particular at the level of the characteristic electron transfer chain, which is limited to merely two tryptophans, instead of the "classical" three or four. Using transient absorption spectroscopy, we show that the dynamics of photoreduction of the oxidized FAD cofactor in the NewPHL begins similarly as that in canonical photolyases, i.e., with a sub-ps primary reduction of the excited FAD cofactor by an adjacent tryptophan, followed by migration of the electron hole towards the second tryptophan in the tens of ps regime. However, the resulting tryptophanyl radical then undergoes an unprecedentedly fast deprotonation in less than 100 ps in the NewPHL. In spite of the stabilization effect of this deprotonation, almost complete charge recombination follows in two phases of ~ 950 ps and ~ 50 ns. Such a rapid recombination of the radical pair implies that the first FAD photoreduction step, i.e., conversion of the fully oxidized to the semi-quinone state, should be rather difficult in vivo. We hence suggest that the flavin chromophore likely switches only between its semi-reduced and fully reduced form in NewPHL under physiological conditions.

A topologically distinct class of photolyases specific for UV lesions within single-stranded DNA.

Photolyases are ubiquitously occurring flavoproteins for catalyzing photo repair of UV-induced DNA damages. All photolyases described so far have a bilobal architecture with a C-terminal domain comprising flavin adenine dinucleotide (FAD) as catalytic cofactor and an N-terminal domain capable of harboring an additional antenna chromophore. Using sequence-similarity network analysis we discovered a novel subgroup of the photolyase/cryptochrome superfamily (PCSf), the NewPHLs. NewPHL occur in bacteria and have an inverted topology with an N-terminal catalytic domain and a C-terminal domain for sealing the FAD binding site from solvent access. By characterizing two NewPHL we show a photochemistry characteristic of other PCSf members as well as light-dependent repair of CPD lesions. Given their common specificity towards single-stranded DNA many bacterial species use NewPHL as a substitute for DASH-type photolyases. Given their simplified architecture and function we suggest that NewPHL are close to the evolutionary origin of the PCSf.

Immunofluorescent distribution of postribosomal particles during oogenesis and early development of an insect (Dysdercus intermedius, Heteroptera, Pyrrhoc.).

During previtellogenesis, the oocytes of the telotrophic meroistic ovary ofDysdercus are provided with ribosomes and ribonucleoprotein (RNP) particles by the nurse cells. At the end of vitellogenesis, the oocyte itself becomes active as shown by autoradiography. The proteins synthesized by the oocyte are stored in cytoplasmic postribosomal particles which are preformed by the tropharium. The proteins of these particles were separated by SDS polyacrylamide gels and their endogenous oocyte proteins revealed by fluorography. The synthesis, transport, and storage of the postribosomal particles are demonstrated by indirect immunofluorescence. The young oocytes of previtellogenic follicles show a diffuse distribution of these particles. In late vitellogenesis, fluorescence becomes more and more concentrated in spots throughout a distinct region in the middle part of the oocyte. Thus, in freshly laid eggs, the periplasm is free of fluorescence. During migration of the cleavage nuclei the postribosomal particles were shifted into the cortex. Fluorescence is then most intense in the periplasmic region. During blastoderm formation, however, fluorescence decreases.

Properties of two follicle proteins and their possible role for vitellogenesis in the African Locust.

Insoluble proteins from the maturing follicle ofLocusta migratoria were analyzed by SDS-PAGE. A reproducible pattern of low molecular weight proteins was observed. Five of these proteins did not correspond to yolk or haemolymph proteins. At least two of these show marked quantitative changes during oocyte development. By in vitro incubation of follicles and fat body with a labelled precursor, and by the identification of the labelled polypeptides by SDS-PAGE, we could demonstrate that these two proteins are synthesized only during the time of vitellogenin uptake. This protein is probably a follicle product necessary for yolk formation. The other protein might be necessary for vitelline membrane and/or chorion formation.

Developmental studies on two ecdysone deficient mutants ofDrosophila melanogaster.

This paper describes two ecdysone-deficient, recessive-lethal mutants,lethal(1)giant ring gland (grg) andlethal(1)suppressor of forked mad-ts (mad-ts: Jürgens and Gateff 1979) and compares their ecdysteroid titers with that of the wild-type. Mutant larvae show a much reduced ecdysteroid content, amounting to 1/10 to 1/30 of the wild-type values, but never a true titer peak. They fail to pupate and die after 1-3 weeks. Ecdysteroid feeding elicits different responses in the larvae of the two mutants.mad-ts larvae pupate within 24 h, thus showing that their low ecdysteroid titer is directly connected to their inability to pupate.mad-ts resembles the mutantlethal (3)ecdysone-1 ts (Garen et al. 1977). Thegrg mutant larvae, on the other hand, fail to pupate after 20-hydroxyecdysone feeding as well as injection. The primary defect of thegrg mutant is not entirely clear. Thegrg larval salivary gland cells appear to possess normal ecdysteroid receptors. Furthermore, the low ecdysteroid titer ingrg is not the result of an increased ecdysteroid catabolism. The primary defect in the mutant may lie in the malfunctioning neurosecretory cells which do not show neurosecretion in histological preparations. Further support for this notion comes from electronmicrographs of the enlargedgrg ring glands which, in contrast to the wild-type, do not possess nerve endings.In the wild-type three ecdysteroid peaks were found: one shortly before puparium formation, the second at approximately 12 h and the third at about 30 h after pupation. The ecdysteroid titer peak in late third instar, wild-type larvae is mainly due to the presence of 20-dydroxyecdysone as shown by radioimmunoassays after thin layer chromatography and derivatization followed by gas liquid chromatography and mass spectroscopy. In addition, a number of unidentified polar and apolar metabolites were also present.