Crystal Structures of Bacterial (6‐4) Photolyase Mutants with Impaired DNA Repair Activity

PhrB from Agrobacterium fabrum is the first prokaryotic photolyase which repairs (6‐4) UV DNA photoproducts. The protein harbors three cofactors: the enzymatically active FAD chromophore, a second chromophore, 6,7‐dimethyl‐8‐ribityllumazine (DMRL) and a cubane‐type Fe‐S cluster. Tyr424 of PhrB is part of the DNA‐binding site and could provide an electron link to the Fe‐S cluster. The PhrB_Y424F mutant showed reduced binding of lesion DNA and loss of DNA repair. The mutant PhrB_I51W is characterized by the loss of the DMRL chromophore, reduced photoreduction and reduced DNA repair capacity. We have determined the crystal structures of both mutants and found that both mutations only affect local protein environments, whereas the overall fold remained unchanged. The crystal structure of PhrB_Y424F revealed a water network extending to His366, which are part of the lesion‐binding site. The crystal structure of PhrB_I51W shows how the bulky Trp leads to structural rearrangements in the DMRL chromophore pocket. Spectral characterizations of PhrB_I51W suggest that DMRL serves as an antenna chromophore for photoreduction and DNA repair in the wild type. The energy transfer from DMRL to FAD could represent a phylogenetically ancient process.     

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/β-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.     

Identification and Purification of the CPD Photolyase in Vibrio parahaemolyticus RIMD2210633

Photoreactivation is an error‐free mechanism of DNA repair, utilized by prokaryotes and most eukaryotes and is catalyzed by specific enzymes called DNA photolyases. Photoreactivation has been reported in Vibrio parahaemolyticus WP28; however, information on photolyases in V. parahaemolyticus (V.p) strains has not been reported. This study examined the photoreactivation in V.p RIMD2210633. The photolyase responsible for repairing cyclobutane pyrimidine dimer (CPD) in DNA was identified, and the corresponding gene was determined as VPA1471. The protein was overexpressed in Escherichia coli and was purified for functional assessment in vitro. The mRNA level and protein expression level of this gene increased after ultraviolet A (UVA) illumination following ultraviolet C (UVC) irradiation. In vitro experiments confirmed that the protein encoded by VPA1471 could reduce the quantity of CPD in DNA. We designated the corresponding gene and protein of VPA1471 phr and Phr, respectively, although the function of two other photolyase/cryptochrome family members, VPA0203 and VPA0204, remains unclear. UV (ultraviolet) irradiation experiments suggest that these two genes possess some photorepairing ability. Therefore, we hypothesize that VPA0203 and VPA0204 encode (6‐4) photolyase in V. parahaemolyticus RIMD2210633.     

Single Amino Acid Substitution Reveals Latent Photolyase Activity in Arabidopsis cry1

A quick switch: A single amino acid substitution at a conserved residue (D396N) of Arabidopsis cryptochrome-1 (Atcry1) confers single-stranded DNA repair activity in?vitro, conferring photolyase activity onto the cryptochrome. The mutant protein undergoes photoreduction of flavin to the fully reduced anionic form, similar to photolyases and unlike wild-type cryptochromes.     

Metal‐Ion‐Mediated Tuning of Duplex DNA Binding by Bis(2‐(2‐pyridyl)‐1H‐benzimidazole)

Studies of double‐stranded‐DNA binding have been performed with three isomeric bis(2‐(n‐pyridyl)‐1H‐benzimidazole)s (n=2, 3, 4). Like the well‐known Hoechst 33258, which is a bisbenzimidazole compound, these three isomers bind to the minor groove of duplex DNA. DNA binding by the three isomers was investigated in the presence of the divalent metal ions Mg²⁺, Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺. Ligand–DNA interactions were probed with fluorescence and circular dichroism spectroscopy. These studies revealed that the binding of the 2‐pyridyl derivative to DNA is dramatically reduced in the presence of Co²⁺, Ni²⁺, and Cu²⁺ ions and is abolished completely at a ligand/metal‐cation ratio of 1:1. Control experiments done with the isomeric 3‐ and 4‐pyridyl derivatives showed that their binding to DNA is unaffected by the aforementioned transition‐metal ions. The ability of 2‐(2‐pyridyl)benzimidazole to chelate metal ions and the conformational changes of the ligand associated with ion chelation probably led to such unusual binding results for the ortho isomer. The addition of ethylenediaminetetraacetic acid (EDTA) reversed the effects completely.     

Targeted Inactivation of DNA Photolyase Genes in Medaka Fish (Oryzias latipes)

Proteins of the cryptochrome/photolyase family (CPF) exhibit sequence and structural conservation, but their functions are divergent. Photolyase is a DNA repair enzyme that catalyzes the light‐dependent repair of ultraviolet (UV)‐induced photoproducts, whereas cryptochrome acts as a photoreceptor or circadian clock protein. Two types of DNA photolyase exist: CPD photolyase, which repairs cyclobutane pyrimidine dimers (CPDs), and 6‐4 photolyase, which repairs 6‐4 pyrimidine–pyrimidone photoproducts (6‐4PPs). Although the Cry‐DASH protein is classified as a cryptochrome, it also has light‐dependent DNA repair activity. To determine the significance of the three light‐dependent repair enzymes in recovering from solar UV‐induced DNA damage at the organismal level, we generated mutants in each gene in medaka using the CRISPR genome editing technique. The light‐dependent repair activity of the mutants was examined in vitro in cultured cells and in vivo in skin tissue. Light‐dependent repair of CPD was lost in the CPD photolyase‐deficient mutant, whereas weak repair activity against 6‐4PPs persisted in the 6‐4 photolyase‐deficient mutant. These results suggest the existence of a heretofore unknown 6‐4PP repair pathway and thus improve our understanding of the mechanisms of defense against solar UV in vertebrates.     

Repair of (6‐4) Lesions in DNA by (6‐4) Photolyase: 20 Years of Quest for the Photoreaction Mechanism

Exposure of DNA to ultraviolet (UV) light from the Sun or from other sources causes the formation of harmful and carcinogenic crosslinks between adjacent pyrimidine nucleobases, namely cyclobutane pyrimidine dimers and pyrimidine(6–4)pyrimidone photoproducts. Nature has developed unique flavoenzymes, called DNA photolyases, that utilize blue light, that is photons of lower energy than those of the damaging light, to repair these lesions. In this review, we focus on the chemically challenging repair of the (6–4) photoproducts by (6–4) photolyase and describe the major events along the quest for the reaction mechanisms, over the 20 years since the discovery of (6‐4) photolyase.     

Light-induced conformational change and product release in DNA repair by (6-4) photolyase

Proteins of the cryptochrome/photolyase family share high sequence similarities, common folds, and the flavin adenine dinucleotide (FAD) cofactor, but exhibit diverse physiological functions. Mammalian cryptochromes are essential regulatory components of the 24 h circadian clock, whereas (6-4) photolyases recognize and repair UV-induced DNA damage by using light energy absorbed by FAD. Despite increasing knowledge about physiological functions from genetic analyses, the molecular mechanisms and conformational dynamics involved in clock signaling and DNA repair remain poorly understood. The (6-4) photolyase, which has strikingly high similarity to human clock cryptochromes, is a prototypic biological system to study conformational dynamics of cryptochrome/photolyase family proteins. The entire light-dependent DNA repair process for (6-4) photolyase can be reproduced in a simple in vitro system. To decipher pivotal reactions of the common FAD cofactor, we accomplished time-resolved measurements of radical formation, diffusion, and protein conformational changes during light-dependent repair by full-length (6-4) photolyase on DNA carrying a single UV-induced damage. The (6-4) photolyase by itself showed significant volume changes after blue-light activation, indicating protein conformational changes distant from the flavin cofactor. A drastic diffusion change was observed only in the presence of both (6-4) photolyase and damaged DNA, and not for (6-4) photolyase alone or with undamaged DNA. Thus, we propose that this diffusion change reflects the rapid (50 μs time constant) dissociation of the protein from the repaired DNA product. Conformational changes with such fast turnover would likely enable DNA repair photolyases to access the entire genome in cells.     

An Ethenoadenine FAD Analog Accelerates UV Dimer Repair by DNA Photolyase

Reduced anionic flavin adenine dinucleotide (FADH^?) is the critical cofactor in DNA photolyase (PL) for the repair of cyclobutane pyrimidine dimers (CPD) in UV‐damaged DNA. The initial step involves photoinduced electron transfer from *FADH^? to the CPD. The adenine (Ade) moiety is nearly stacked with the flavin ring, an unusual conformation compared to other FAD‐dependent proteins. The role of this proximity has not been unequivocally elucidated. Some studies suggest that Ade is a radical intermediate, but others conclude that Ade modulates the electron transfer rate constant (k_ET) through superexchange. No study has succeeded in removing or modifying this Ade to test these hypotheses. Here, FAD analogs containing either an ethano‐ or etheno‐bridged Ade between the AN1 and AN6 atoms (e‐FAD and ε‐FAD, respectively) were used to reconstitute apo‐PL, giving e‐PL and ε‐PL respectively. The reconstitution yield of e‐PL was very poor, suggesting that the hydrophobicity of the ethano group prevented its uptake, while ε‐PL showed 50% reconstitution yield. The substrate binding constants for ε‐PL and rPL were identical. ε‐PL showed a 15% higher steady‐state repair yield compared to FAD‐reconstituted photolyase (rPL). The acceleration of repair in ε‐PL is discussed in terms of an ε‐Ade radical intermediate vs superexchange mechanism.     

DNA光解酶模型研究的近期进展

用模型化合物来模拟DNA光解酶与底物的作用, 有助于认识DNA光复活作用机理. 评述了环丁烷型嘧啶二聚体光解酶和(6-4)光产物光解酶的模型研究进展, 并展望了该领域的发展前景．     

Solid‐state nuclear magnetic resonance investigation of neurosteroid compounds and magnesium interactions

The neurosteroid trans‐dehydroandrosterone (DHEA) and its analogs with slightly different modifications in the side chain attached to C17, that is, (3S)‐acetoxypregn‐5‐en‐20‐one ( 1 ) and (3S,20R)‐acetoxypregn‐5‐en‐20‐ol ( 2 ), have been synthesized to investigate DHEA–cation interactions. In this study, we applied solid‐state ¹H/¹³C cross‐polarization/magic‐angle spinning (CP/MAS) nuclear magnetic resonance (NMR) spectroscopy to a series of DHEA analog/Mg²⁺ mixtures at different Mg²⁺ concentrations. The high‐resolution ¹³C NMR spectra of 1 /Mg²⁺ mixtures exhibit two distinct ¹³C spectral patterns, one attributable to 1 free from Mg²⁺, and the other attributable to 1 with bound Mg²⁺. For 2 , the ¹³C NMR spectra exhibit three distinct spectral patterns; besides that of the free form, the other two can be assigned to Mg²⁺‐bound forms. Based on the analysis of the chemical shift deviations (CSDs), we conclude that both 1 and 2 might be subject to a cation–π interaction via the C5–C6 double bond, in contrast to that observed previously for DHEA. As demonstrated, DHEA possesses two Mg²⁺ binding sites, that is, C17–O and C5–C6 double bond, in which the binding affinity of the former is at least three times stronger than that of the latter. The solid‐state ¹³C NMR investigation allows better understanding of the underlying cation binding effects of neurosteroid molecules in vitro.     

On the Stability of DNA Origami Nanostructures in Low‐Magnesium Buffers

DNA origami structures have great potential as functional platforms in various biomedical applications. Many applications, however, are incompatible with the high Mg²⁺ concentrations commonly believed to be a prerequisite for maintaining DNA origami integrity. Herein, we investigate DNA origami stability in low‐Mg²⁺ buffers. DNA origami stability is found to crucially depend on the availability of residual Mg²⁺ ions for screening electrostatic repulsion. The presence of EDTA and phosphate ions may thus facilitate DNA origami denaturation by displacing Mg²⁺ ions from the DNA backbone and reducing the strength of the Mg²⁺–DNA interaction, respectively. Most remarkably, these buffer dependencies are affected by DNA origami superstructure. However, by rationally selecting buffer components and considering superstructure‐dependent effects, the structural integrity of a given DNA origami nanostructure can be maintained in conventional buffers even at Mg²⁺ concentrations in the low‐micromolar range.     

Mg2+‐Dependent High Mechanical Anisotropy of Three‐Way‐Junction pRNA as Revealed by Single‐Molecule Force Spectroscopy

Mechanical anisotropy is ubiquitous in biological tissues but is hard to reproduce in synthetic biomaterials. Developing molecular building blocks with anisotropic mechanical response is the key towards engineering anisotropic biomaterials. The three‐way‐junction (3WJ) pRNA, derived from ϕ 29 DNA packaging motor, shows strong mechanical anisotropy upon Mg²⁺ binding. In the absence of Mg²⁺, 3WJ‐pRNA is mechanically weak without noticeable mechanical anisotropy. In the presence of Mg²⁺, the unfolding forces can differ by more than 4‐fold along different pulling directions, ranging from about 47 pN to about 219 pN. Mechanical anisotropy of 3WJ‐pRNA stems from pulling direction dependent cooperativity for the rupture of two Mg²⁺ binding sites, which is a novel mechanism for the mechanical anisotropy of biomacromolecules. It is anticipated that 3WJ‐pRNA can be used as a key element for the construction of biomaterials with controllable mechanical anisotropy.     

Enzyme‐Mediated Endogenous and Bioorthogonal Control of a DNAzyme Fluorescent Sensor for Imaging Metal Ions in Living Cells

Bioorthogonal control of metal‐ion sensors for imaging metal ions in living cells is important for understanding the distribution and fluctuation of metal ions. Reported here is the endogenous and bioorthogonal activation of a DNAzyme fluorescent sensor containing an 18‐base pair recognition site of a homing endonuclease (I‐SceI), which is found by chance only once in 7×10¹⁰ bp of genomic sequences, and can thus form a near bioorthogonal pair with I‐SceI for DNAzyme activation with minimal effect on living cells. Once I‐SceI is expressed inside cells, it cleaves at the recognition site, allowing the DNAzyme to adopt its active conformation. The activated DNAzyme sensor is then able to specifically catalyze cleavage of a substrate strand in the presence of Mg²⁺ to release the fluorophore‐labeled DNA fragment and produce a fluorescent turn‐on signal for Mg²⁺. Thus I‐SceI bioorthogonally activates the 10–23 DNAzyme for imaging of Mg²⁺ in HeLa cells.     

A Review of Spectroscopic and Biophysical‐Chemical Studies of the Complex of Cyclobutane Pyrimidine Dimer Photolyase and Cryptochrome DASH with Substrate DNA

Cyclobutane pyrimidine dimer (CPD) photolyase (PL) is a structure‐specific DNA repair enzyme that uses blue light to repair CPD on DNA. Cryptochrome (CRY) DASH enzymes use blue light for the repair of CPD lesions on single‐stranded (ss) DNA, although some may also repair these lesions on double‐stranded (ds) DNA. In addition, CRY DASH may be involved in blue light signaling, similar to cryptochromes. The focus of this review is on spectroscopic and biophysical‐chemical experiments of the enzyme–substrate complex that have contributed to a more detailed understanding of all the aspects of the CPD repair mechanism of CPD photolyase and CRY DASH. This will be performed in the backdrop of the available X‐ray crystal structures of these enzymes bound to a CPD‐like lesion. These structures helped to confirm conclusions that were drawn earlier from spectroscopic and biophysical‐chemical experiments, and they have a critical function as a framework to design new experiments and to interpret new experimental data. This review will show the important synergy between X‐ray crystallography and spectroscopic/biophysical‐chemical investigations that is essential to obtain a sufficiently detailed picture of the overall mechanism of CPD photolyases and CRY DASH proteins.     

Nicotinamide Adenine Dinucleotides Arrest Photoreduction of Class II DNA Photolyases in FADH˙ State

All light‐sensitive members of the photolyase/cryptochrome family rely on FAD as catalytic cofactor. Its activity is regulated by photoreduction, a light‐triggered electron transfer process from a conserved tryptophan triad to the flavin. The stability of the reduced flavin depends on available external electron donors and oxygen. In this study, we show for the class II photolyase of Methanosarcina mazei , Mm CPDII , that it utilizes physiologically relevant redox cofactors NADH and NADPH for the formation of the semiquinoid FAD in a light‐dependent reaction. Using redox‐inert variants Mm CPDII /W388F and Mm CPDII /W360F, we demonstrate that photoreduction by NADH and NADPH requires the class II ‐specific tryptophan cascade of Mm CPDII . Finally, we confirmed that mutations in the tryptophan cascade can be introduced without any substantial structural disturbances by analyzing crystal structures of Mm CPDII /W388F, Mm CPDII /W360F and Mm CPDII /Y345F.     

Amphiphilic Schiff Base Organogels: Metal‐Ion‐Mediated Chiral Twists and Chiral Recognition

New amphiphilic gelators that contained both Schiff base and L ‐glutamide moieties, abbreviated as o‐SLG and p‐SLG, were synthesized and their self‐assembly in various organic solvents in the absence and presence of metal ions was investigated. Gelation test revealed that o‐SLG formed a thermotropic gel in many organic solvents, whilst p‐SLG did not. When metal ions, such as Cu²⁺, Zn²⁺, Mg²⁺, Ni²⁺, were added, different behaviors were observed. The addition of Cu²⁺ induced p‐SLG to from an organogel. In the case of o‐SLG, the addition of Cu²⁺ and Mg²⁺ ions maintained the gelating ability of the compound, whilst Zn²⁺ and Ni²⁺ ions destroyed the gel. In addition, the introduction of Cu²⁺ ions caused the nanofiber gel to perform a chiral twist, whilst the Mg²⁺ ions enhanced the fluorescence of the gel. More interestingly, the Mg²⁺‐ion‐mediated organogel showed differences in the fluorescence quenching by D ‐ and L ‐tartaric acid, thus showing a chiral recognition ability.     

Photoreactivating Enzyme for (6–4) Photoproducts in Cultured Goldfish Cells

We previously reported that when cultured goldfish cells are illuminated with fluorescent light, photorepair ability for both cyclobutane pyrimidine dimers and (6–4) photoproducts increased. In the present study, it was found that the duration of the induced photorepair ability for cyclobutane pyrimidine dimers was longer than that for (6–4) photoproducts, suggesting the presence of different photolyases for repair of these two major forms of DNA damage. A gel shift assay was then performed to show the presence of protein(s) binding to (6–4) photoproducts and its dissociation from (6–4) photoproducts under fluorescent light illumination. In addition, at 8 h after fluorescent light illumination of the cell, the binding of pro-tein(s) to (6–4) photoproducts increased. The restriction enzymes that have recognition sites containing TT or TC sequences failed to digest the UV-irradiated DNA pho-toreactivated by using Escherichia coli photolyase for cyclobutane pyrimidine dimers, indicating that restriction enzymes could not function because (6–4) photoproducts remained in recognition sites. But, when UV-irradiated DNA depleted of cyclobutane pyrimidine dimers was incubated with extract of cultured goldfish cells under fluorescent light illumination, it was digested with those restriction enzymes. These results suggested the presence of (6–4) photolyase in cultured goldfish cells as in Dro-sophila, Xenopus and Crotalus.