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.

Filming DNA repair at the atomic level.

Dissection of multistep catalysis by a photoenzyme could inspire green chemistry applications.

UV-driven self-repair of cyclobutane pyrimidine dimers in RNA.

Nucleic acids can be damaged by ultraviolet (UV) irradiation, forming structural photolesions such as cyclobutane-pyrimidine-dimers (CPD). In modern organisms, sophisticated enzymes repair CPD lesions in DNA, but to our knowledge, no RNA-specific enzymes exist for CPD repair. Here, we show for the first time that RNA can protect itself from photolesions by an intrinsic UV-induced self-repair mechanism. This mechanism, prior to this study, has exclusively been observed in DNA and is based on charge transfer from CPD-adjacent bases. In a comparative study, we determined the quantum yields of the self-repair of the CPD-containing RNA sequence, GAU = U to GAUU (0.23%), and DNA sequence, d(GAT = T) to d(GATT) (0.44%), upon 285 nm irradiation via UV/Vis spectroscopy and HPLC analysis. After several hours of irradiation, a maximum conversion yield of ∼16% for GAU = U and ∼33% for d(GAT = T) was reached. We examined the dynamics of the intermediate charge transfer (CT) state responsible for the self-repair with ultrafast UV pump - IR probe spectroscopy. In the dinucleotides GA and d(GA), we found comparable quantum yields of the CT state of ∼50% and lifetimes on the order of several hundred picoseconds. Charge transfer in RNA strands might lead to reactions currently not considered in RNA photochemistry and may help understanding RNA damage formation and repair in modern organisms and viruses. On the UV-rich surface of the early Earth, these self-stabilizing mechanisms likely affected the selection of the earliest nucleotide sequences from which the first organisms may have developed.

Mapping the recognition pathway of cyclobutane pyrimidine dimer in DNA by Rad4/XPC.

UV radiation-induced DNA damages have adverse effects on genome integrity and cellular function. The most prevalent UV-induced DNA lesion is the cyclobutane pyrimidine dimer (CPD), which can cause skin disorders and cancers in humans. Rad4/XPC is a damage sensing protein that recognizes and repairs CPD lesions with high fidelity. However, the molecular mechanism of how Rad4/XPC interrogates CPD lesions remains elusive. Emerging viewpoints indicate that the association of Rad4/XPC with DNA, the insertion of a lesion-sensing ß-hairpin of Rad4/XPC into the lesion site and the flipping of CPD's partner bases (5'-dA and 3'-dA) are essential for damage recognition. Characterizing these slow events is challenging due to their infrequent occurrence on molecular time scales. Herein, we have used enhanced sampling and molecular dynamics simulations to investigate the mechanism and energetics of lesion recognition by Rad4/XPC, considering multiple plausible pathways between the crystal structure of the Rad4-DNA complex and nine intermediate states. Our results shed light on the most likely sequence of events, their potential coupling and energetics. Upon association, Rad4 and DNA form an encounter complex in which CPD and its partner bases remain in the duplex and the BHD3 ß-hairpin is yet to be inserted into the lesion site. Subsequently, sequential base flipping occurs, with the flipping of the 5'-dA base preceding that of the 3'-dA base, followed by the insertion of the BHD3 ß-hairpin into the lesion site. The results presented here have significant implications for understanding the molecular basis of UV-related skin disorders and cancers and for paving the way for novel therapeutic strategies.

Tracking the Early Stage of Triplet-Induced Photodamage in a DNA Dimer and Oligomer Containing 5-Methylcytosine.

Methylation at the C5 position of cytosine, a naturally occurring epigenetic modification on DNA, shows a high correlation with mutational hotspots in disease such as skin cancer. Due to its essential biological relevance, numerous studies were devoted to confirming that the methylated sites favor the formation of the cyclobutane pyrimidine dimer (CPD), a well-known UV-induced lesion. However, photophysical and photochemical properties of dinucleotides and polynucleotides containing 5-methylcytosine (5mC) remain elusive. Herein, a charge transfer (CT) triplet state, generated via intersystem crossing (ISC) from a CT singlet state that enhanced after methylation on cytosine, is directly observed by using femtosecond transient absorption (TA) and time-resolved mid-infrared (TRIR) spectroscopy together with quantum chemical calculations for the first time in the T5mC dimer. Such an ISC process is quenched due to limitations of the ground-state geometries in 5mC-containing single-strand oligomer d(T5mC)9. This mechanistic information is important for understanding the early stage of triplet state-induced CPD formation in 5mC containing DNA.

Reductive Photocycloreversion of Cyclobutane Dimers Triggered by Guanines.

The quest for simple systems achieving the photoreductive splitting of four-membered ring compounds is a matter of interest not only in organic chemistry but also in biochemistry to mimic the activity of DNA photorepair enzymes. In this context, 8-oxoguanine, the main oxidatively generated lesion of guanine, has been shown to act as an intrinsic photoreductant by transferring an electron to bipyrimidine lesions and provoking their cycloreversion. But, in spite of appropriate photoredox properties, the capacity of guanine to repair cyclobutane pyrimidine dimer is not clearly established. Here, dyads containing the cyclobutane thymine dimer and guanine or 8-oxoguanine are synthesized, and their photoreactivities are compared. In both cases, the splitting of the ring takes place, leading to the formation of thymine, with a quantum yield 3.5 times lower than that for the guanine derivative. This result is in agreement with the more favored thermodynamics determined for the oxidized lesion. In addition, quantum chemistry calculations and molecular dynamics simulations are carried out to rationalize the crucial aspects of the overall cyclobutane thymine dimer photoreductive repair triggered by the nucleobase and its main lesion.

Post- and Pre-Radiolabeling Assays for anti Thymidine Cyclobutane Dimers as Intrinsic Photoprobes of Various Types of G-Quadruplexes, Reverse Hoogsteen Hairpins, and Other Non-B DNA Structures.

G-quadruplexes are thought to play an important role in gene regulation and telomere maintenance, but developing probes for their presence and location is challenging due to their transitory and highly dynamic nature. The majority of probes for G-quadruplexes have relied on antibody or small-molecule binding agents, many of which can also alter the dynamics and relative populations of G-quadruplexes. Recently, it was discovered that ultraviolet B (UVB) irradiation of human telomeric DNA and various G-quadruplex forming sequences found in human promoters, as well as reverse Hoogsteen hairpins, produces a unique class of non-adjacent anti cyclobutane pyrimidine dimers (CPDs). Therefore, one can envision using a pulse of UVB light to irreversibly trap these non-B DNA structures via anti CPD formation without perturbing their dynamics, after which the anti CPDs can be identified and mapped. As a first step toward this goal, we report radioactive post- and pre-labeling assays for the detection of non-adjacent CPDs and illustrate their use in detecting trans,anti T=(T) CPD formation in a human telomeric DNA sequence. Both assays make use of snake venom phosphodiesterase (SVP) to degrade the trans,anti T=(T) CPD-containing DNA to the tetranucleotide pTT=(pTT) corresponding to CPD formation between the underlined T's of two separate dinucleotides while degrading the adjacent syn TT CPDs to the trinucleotide pGT=T. In the post-labeling assay, calf intestinal phosphodiesterase is used to dephosphorylate the tetranucleotides, which are then rephosphorylated with kinase and [32P]-ATP to produce radiolabeled mono- and diphosphorylated tetranucleotides. The tetranucleotides are confirmed to be non-adjacent CPDs by 254 nm photoreversion to the dinucleotide p*TT. In the pre-labeling assay, radiolabeled phosphates are introduced into non-adjacent CPD-forming sites by ligation prior to irradiation, thereby eliminating the dephosphorylation and rephosphorylation steps. The assays are also demonstrated to detect the stereoisomeric cis,anti T=(T) CPD.

Role of Substrate Binding Interactions on DNA Repair by Photolyase.

The repair of the cyclobutane pyrimidine dimer (CPD) lesion in DNA by photolyase is determined by its initial recognition, and the catalytic efficiency depends on a series of intermolecular electron-transfer (ET) processes. Here, we investigated the repair of a CPD structural isomer, replacing the deoxyribose with a pyranose sugar on the 5' site, and found a loss in binding efficiency and repair quantum yield. Using femtosecond spectroscopy, we characterized all elementary repair steps and observed a systemic slowdown of the four intermolecular ET reactions and the second bond splitting. Our observations and molecular dynamics simulations suggest that the sugar replacement disrupts the lesion binding configuration, weakening the electronic coupling between the cofactor and lesion and altering the stability of lesion intermediates. These findings highlight how the CPD photolyases have utilized the structural features of the CPD lesion and optimized its interactions with the cofactor and key active-site residues to maximize repair yields.

Coupling of conformation and CPD damage in nucleosomal DNA.

UV-light can cause photodimerization and hence damages in DNA. Most frequent are cyclobutane pyrimidine dimer (CPD) damages, which predominantly form at TpT (thymine-thymine) steps. It is well known that CPD damage probability is different for single-stranded or double stranded DNA and depends on the sequence context. However, DNA deformation due to packing in nucleosomes can also influence CPD formation. Quantum mechanical calculations and Molecular Dynamics simulations indicate little CPD damage probability for DNA's equilibrium structure. We find that DNA needs to be deformed in a specific way to allow the HOMO → LUMO transition required for CPD damage formation. The simulation studies further show that the periodic CPD damage patterns measured in chromosomes and nucleosomes can be directly explained by the periodic deformation pattern of the DNA in the nucleosome complex. It supports previous findings on characteristic deformation patterns found in experimental nucleosome structures that relate to CPD damage formation. The result may have important implications for our understanding of UV-induced DNA mutations in human cancers.

Chemiexcited Neurotransmitters and Hormones Create DNA Photoproducts in the Dark.

In DNA, electron excitation allows adjacent pyrimidine bases to dimerize by [2 + 2] cycloaddition, creating chemically stable but lethal and mutagenic cyclobutane pyrimidine dimers (CPDs). The usual cause is ultraviolet radiation. Alternatively, CPDs can be made in the dark (dCPDs) via chemically mediated electron excitation of the skin pigment melanin, after it is oxidized by peroxynitrite formed from the stress-induced radicals superoxide and nitric oxide. We now show that the dark process is not limited to the unusual structural molecule melanin: signaling biomolecules such as indolamine and catecholamine neurotransmitters and hormones can also be chemiexcited to energy levels high enough to form dCPDs. Oxidation of serotonin, dopamine, melatonin, and related biogenic amines by peroxynitrite created triplet-excited species, evidenced by chemiluminescence, energy transfer to a triplet-state reporter, or transfer to O2 resulting in singlet molecular oxygen. For a subset of these signaling molecules, triplet states created by peroxynitrite or peroxidase generated dCPDs at levels comparable to ultraviolet (UV). Neurotransmitter catabolism by monoamine oxidase also generated dCPDs. These results reveal a large class of signaling molecules as electronically excitable by biochemical reactions and thus potential players in deviant mammalian metabolism in the absence of light.

Adjacent and Nonadjacent Dipyrimidine Photoproducts as Intrinsic Probes of DNA Secondary and Tertiary Structure†.

While the photochemistry of duplex DNA has been extensively studied, the photochemistry of nonduplex DNA structures is largely unexplored. Because the structure and stereochemistry of DNA photoproducts depend on the secondary structure and conformation of the DNA precursor, they can serve as intrinsic probes of DNA structure. This review focuses on the structures and stereoisomers of pyrimidine dimer photoproducts arising from adjacent and nonadjacent pyrimidines in A, B and denatured DNA, bulge loops, G-quadruplexes and reverse Hoogsteen hairpins and methods for their detection.

Heliorhodopsin Helps Photolyase to Enhance the DNA Repair Capacity.

Light quality is a significant factor for living organisms that have photosensory systems, such as rhodopsin, a seven alpha-helical transmembrane protein with the retinal chromophore. Here, we report, for the first time, the function of new rhodopsin, which is an inverted 7-transmembrane protein, isolated from Trichococcus flocculiformis. T. flocculiformis heliorhodopsin (TfHeR) works as a regulatory helper rhodopsin that binds with class 2 cyclobutane pyrimidine dimer (CPDII) photolyase to broaden the spectrum and upregulate DNA repair activity. We have confirmed their interaction through isothermal titration calorimetry (dissociation constant of 21.7 µM) and identified the charged residues for the interaction. Based on in vivo and in vitro experiments, we showed that the binding of heliorhodopsin with photolyase improved photolyase activity by about 3-fold to repair UV-caused DNA damage. Also, the DNA repair activity of TfHeR/T. flocculiformis photolyase (TfPHR) was observed in the presence of green light. Our results suggested that heliorhodopsin directly controls the activity of photolyase and coevolves to broaden the activity spectrum by protein-protein interaction. IMPORTANCE This study reports a function for Heliorhodopsin working as a regulatory helper rhodopsin that with CPDII photolyase to broaden the spectrum and upregulating the DNA repair activity. Our results suggested that heliorhodopsin directly controls photolyase activity and coevolves to broaden the DNA repair capacity by protein-protein interaction.

DNA photoproducts released by repair in biological fluids as biomarkers of the genotoxicity of UV radiation.

UV-induced formation of photoproducts in DNA is a major initiating event of skin cancer. Consequently, many analytical tools have been developed for their quantification in DNA. In the present work, we extended our previous liquid chromatography-mass spectrometry method to the quantification of the short DNA fragments containing photoproducts that are released from cells by the repair machinery. We designed a robust protocol including a solid-phase extraction step (SPE), an enzymatic treatment aimed at releasing individual photoproducts, and a liquid chromatography method combining on-line SPE and ultra-high-performance liquid chromatography for optimal specificity and sensitivity. We also added relevant internal standards for a better accuracy. The method was validated for linearity, repeatability, and reproducibility. The limits of detection and quantification were found to be in the fmol range. The proof of concept of the use of excreted DNA repair products as biomarkers of the genotoxicity of UV was obtained first in in vitro studies using cultured HaCat cells and ex vivo on human skin explants. Further evidence was obtained from the detection of pyrimidine dimers in the urine of human volunteers collected after recreational exposure in summer. An assay was designed to quantify the DNA photoproducts released from cells within short fragments by the DNA repair machinery. These oligonucleotides were isolated by solid-phase extraction and enzymatically hydrolyzed. The photoproducts were then quantified by on-line SPE combined with UHPLC-MS/MS with isotopic dilution.

Chromatin-remodeling factor BAZ1A/ACF1 targets UV damage sites in an MLL1-dependent manner to facilitate nucleotide excision repair.

Ultraviolet (UV) light irradiation generates pyrimidine dimers on DNA, such as cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts. Such dimers distort the high-order DNA structure and prevent transcription and replication. The nucleotide excision repair (NER) system contributes to resolving this type of DNA lesion. There are two pathways that recognize pyrimidine dimers. One acts on transcribed strands of DNA (transcription-coupled NER), and the other acts on the whole genome (global genome-NER; GG-NER). In the latter case, DNA damage-binding protein 2 (DDB2) senses pyrimidine dimers with several histone modification enzymes. We previously reported that histone acetyltransferase binding to ORC1 (HBO1) interacts with DDB2 and facilitates recruitment of the imitation switch chromatin remodeler at UV-irradiated sites via an unknown methyltransferase. Here, we found that the phosphorylated histone methyltransferase mixed lineage leukemia 1 (MLL1) was maintained at UV-irradiated sites in an HBO1-dependent manner. Furthermore, MLL1 catalyzed histone H3K4 methylation and recruited the chromatin remodeler bromodomain adjacent to zinc finger domain 1A (BAZ1A)/ATP-utilizing chromatin assembly and remodeling factor 1 (ACF1). Depletion of MLL1 suppressed BAZ1A accumulation at UV-irradiated sites and inhibited the removal of CPDs. These data indicate that the DDB2-HBO1-MLL1 axis is essential for the recruitment of BAZ1A to facilitate GG-NER.

On the propensity of formation of cyclobutane dimers in face-to-face and face-to-back uracil stacks in solution.

UV irradiation of RNA leads to the formation of intra- and inter-strand crosslinks of cyclobutane type. Despite the importance of this reaction, relatively little is known about how the mutual orientation of the two bases affects the outcome of the reaction. Here we report a comparative nonadiabatic molecular dynamics study of face-to-back (F2B) and face-to-face (F2F) stacked uracil-water clusters. The computations were performed using the second-order algebraic-diagrammatic-construction (ADC(2)) method. We found that F2B stacked uracil-water clusters either relax non-reactively to the ground state by an ethylenic twist around the CC bond or remain in the lowest nπ* state in which the two bases gradually move away from each other. This finding is consistent with the low propensity for the formation of intra-strand cyclobutane dimers between adjacent RNA bases. On the contrary, in F2F stacked uracil-water clusters, in addition to non-reactive deactivation, we found a pro-reactive deactivation pathway, which may lead to the formation of cyclobutane uracil dimers in the electronic ground state. On a qualitative level, the observed photodynamics of F2F stacked uracil-water clusters explains the greater propensity of RNA to form inter-strand cyclobutane-type crosslinks.

Identification and characterization of a prokaryotic 6-4 photolyase from Synechococcus elongatus with a deazariboflavin antenna chromophore.

Synechococcus elongatus, formerly known as Anacystis nidulans, is a representative species of cyanobacteria. It is also a model organism for the study of photoreactivation, which can be fully photoreactivated even after receiving high UV doses. However, for a long time, only one photolyase was found in S. elongatus that is only able to photorepair UV induced cyclobutane pyrimidine dimers (CPDs) in DNA. Here, we characterize another photolyase in S. elongatus, which belongs to iron-sulfur bacterial cryptochromes and photolyases (FeS-BCP), a subtype of prokaryotic 6-4 photolyases. This photolyase was named SePhrB that could efficiently photorepair 6-4 photoproducts in DNA. Chemical analyses revealed that SePhrB contains a catalytic FAD cofactor and an iron-sulfur cluster. All of previously reported FeS-BCPs contain 6,7-dimethyl-8-ribityllumazine (DMRL) as their antenna chromophores. Here, we first demonstrated that SePhrB possesses 7,8-didemethyl-8-hydroxy-5-deazariboflavin (8-HDF) as an antenna chromophore. Nevertheless, SePhrB could be photoreduced without external electron donors. After being photoreduced, the reduced FAD cofactor in SePhrB was extremely stable against air oxidation. These results suggest that FeS-BCPs are more diverse than expected which deserve further investigation.

The interplay of supercoiling and thymine dimers in DNA.

Thymine dimers are a major mutagenic photoproduct induced by UV radiation. While they have been the subject of extensive theoretical and experimental investigations, questions of how DNA supercoiling affects local defect properties, or, conversely, how the presence of such defects changes global supercoiled structure, are largely unexplored. Here, we introduce a model of thymine dimers in the oxDNA forcefield, parametrized by comparison to melting experiments and structural measurements of the thymine dimer induced bend angle. We performed extensive molecular dynamics simulations of double-stranded DNA as a function of external twist and force. Compared to undamaged DNA, the presence of a thymine dimer lowers the supercoiling densities at which plectonemes and bubbles occur. For biologically relevant supercoiling densities and forces, thymine dimers can preferentially segregate to the tips of the plectonemes, where they enhance the probability of a localized tip-bubble. This mechanism increases the probability of highly bent and denatured states at the thymine dimer site, which may facilitate repair enzyme binding. Thymine dimer-induced tip-bubbles also pin plectonemes, which may help repair enzymes to locate damage. We hypothesize that the interplay of supercoiling and local defects plays an important role for a wider set of DNA damage repair systems.

Lignin Nanoparticles Deliver Novel Thymine Biomimetic Photo-Adducts with Antimelanoma Activity.

We report here the synthesis of novel thymine biomimetic photo-adducts bearing an alkane spacer between nucleobases and characterized by antimelanoma activity against two mutated cancer cell lines overexpressing human Topoisomerase 1 (TOP1), namely SKMEL28 and RPMI7951. Among them, Dewar Valence photo-adducts showed a selectivity index higher than the corresponding pyrimidine-(6-4)-pyrimidone and cyclobutane counterpart and were characterized by the highest affinity towards TOP1/DNA complex as evaluated by molecular docking analysis. The antimelanoma activity of novel photo-adducts was retained after loading into UV photo-protective lignin nanoparticles as stabilizing agent and efficient drug delivery system. Overall, these results support a combined antimelanoma and UV sunscreen strategy involving the use of photo-protective lignin nanoparticles for the controlled release of thymine dimers on the skin followed by their sacrificial transformation into photo-adducts and successive inhibition of melanoma and alert of cellular UV machinery repair pathways.

Anti Regiospecificity in the Photosensitized Cycloaddition of 4-Tetrazolouracil Nucleoside.

The [2 + 2] photocycloaddition of natural pyrimidine nucleobases is devoid of regioselectivity. Although modified pyrimidines have been developed to selectively obtain syn-cyclobutane isomers, the targeted formation of anti-cyclobutane isomers has not been addressed yet. Herein, using NMR analyses and DFT calculations, we demonstrate that the acetone photosensitized excitation of the 4-tetrazolouracil motif in the nucleoside series specifically provides anti-cyclobutane photoproducts in 51% yield. In addition, the cis stereomer formation is preferred over the trans-cyclobutane formation (71:29).

Mechanistic studies of dinucleotide and oligonucleotide model cyclobutane pyrimidine dimer (CPD) DNA lesions under alkaline conditions.

Cyclobutane pyrimidine dimers (CPDs) are the most abundant mutagenic DNA lesions formed in mammalian cells upon exposure to UV-B radiation (280-315 nm) in sunlight. These lesions are thought to be chemically stable and to withstand high concentrations of acids and bases.While earlier investigations of DNA lesions containing saturated pyrimidines have shown that the C4 carbonyl is a potential target of nucleophilic attack, similar reactions with thymine nucleobase model CPDs clearly showed that the cis-syn CPD (major isomer) is stable in the presence of a high concentration of alkali at room temperature. Here is described the alkaline reactivity of these lesions when contained within a dinucleotide CPD model system. Results using cis-syn CPD formed from dinucleotide 5'-TpT-3' combined with [18O]-labelling indicated that CPD undergoes a water addition at the C4=O groups of these now saturated rings. The intermediate formed, however, completely reverts to the starting lesion. Along with confirming the target of water addition within CPD lesions, it was also determined that the two C4 carbonyls present on adjacent saturated pyrimidine rings of the photolesion undergo water exchange at different rates (3' > 5'). Moreover, the difference in reactivity exhibited by these two positions is not limited to a dinucleotide and was observed also in oligonucleotides. Overall, a full understanding of the chemistry of CPD lesions is crucial to our knowledge of naturally-occuring DNA modifications and may lead to further insight into their detection, modification, and biochemical recognition & repair.