Structural and Dynamic Features of the Recognition of 8-oxoguanosine Paired with an 8-oxoG-clamp by Human 8-oxoguanine-DNA Glycosylase.

8-oxoguanine (oxoG) is formed in DNA by the action of reactive oxygen species. As a highly mutagenic and the most common oxidative DNA lesion, it is an important marker of oxidative stress. Human 8-oxoguanine-DNA glycosylase (OGG1) is responsible for its prompt removal in human cells. OGG1 is a bifunctional DNA glycosylase with N-glycosylase and AP lyase activities. Aspects of the detailed mechanism underlying the recognition of 8-oxoguanine among numerous intact bases and its subsequent interaction with the enzyme's active site amino acid residues are still debated. The main objective of our work was to determine the effect (structural and thermodynamic) of introducing an oxoG-clamp in model DNA substrates on the process of 8-oxoG excision by OGG1. Towards that end, we used DNA duplexes modeling OGG1-specific lesions: 8-oxoguanine or an apurinic/apyrimidinic site with either cytidine or the oxoG-clamp in the complementary strand opposite to the lesion. It was revealed that there was neither hydrolysis of the N-glycosidic bond at oxoG nor cleavage of the sugar-phosphate backbone during the reaction between OGG1 and oxoG-clamp-containing duplexes. Possible structural reasons for the absence of OGG1 enzymatic activity were studied via the stopped-flow kinetic approach and molecular dynamics simulations. The base opposite the damage was found to have a critical effect on the formation of the enzyme-substrate complex and the initiation of DNA cleavage. The oxoG-clamp residue prevented the eversion of the oxoG base into the OGG1 active site pocket and impeded the correct convergence of the apurinic/apyrimidinic site of DNA and the attacking nucleophilic group of the enzyme. An obtained three-dimensional model of the OGG1 complex with DNA containing the oxoG-clamp, together with kinetic data, allowed us to clarify the role of the contact of amino acid residues with DNA in the formation of (and rearrangements in) the enzyme-substrate complex.

Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process.

Nucleotide excision repair (NER) is an evolutionarily conserved mechanism that processes helix-destabilizing and/or -distorting DNA lesions, such as UV-induced photoproducts. Here, we investigate the dynamic protein-DNA interactions during the damage recognition step using single-molecule fluorescence microscopy. Quantum dot-labeled Rad4-Rad23 (yeast XPC-RAD23B ortholog) forms non-motile complexes or conducts a one-dimensional search via either random diffusion or constrained motion. Atomic force microcopy analysis of Rad4 with the ß-hairpin domain 3 (BHD3) deleted reveals that this motif is non-essential for damage-specific binding and DNA bending. Furthermore, we find that deletion of seven residues in the tip of ß-hairpin in BHD3 increases Rad4-Rad23 constrained motion at the expense of stable binding at sites of DNA lesions, without diminishing cellular UV resistance or photoproduct repair in vivo. These results suggest a distinct intermediate in the damage recognition process during NER, allowing dynamic DNA damage detection at a distance.

Spacecraft Segment Damage Identification Method Based on Fiber Optic Strain Difference Field Reconstruction and Norm Calculation.

Real-time online identification of spacecraft segment damage is of great significance for realizing spacecraft structural health monitoring and life prediction. In this paper, a damage response characteristic field inversion algorithm based on the differential reconstruction of strain response is proposed to solve the problem of not being able to recognize the small damages of spacecraft structure directly by the strain response alone. Four crack damage location identification methods based on vector norm computation are proposed, which realize online identification and precise location of structural damage events without external excitation by means of spacecraft structural working loads only. A spacecraft segment structural damage monitoring system based on fiber optic grating sensors was constructed, and the average error of damage localization based on the curvature vector 2 norm calculation was 2.58 mm, and the root-mean-square error was 1.98 mm. The results show that the method has superior engineering applicability for on-orbit service environments.

FapydG in the Shadow of OXOdG-A Theoretical Study of Clustered DNA Lesions.

Genetic information, irrespective of cell type (normal or cancerous), is exposed to a range of harmful factors, which can lead to more than 80 different types of DNA damage. Of these, oxoG and FapyG have been identified as the most abundant in normoxic and hypoxic conditions, respectively. This article considers d[AFapyGAOXOGA]*[TCTCT] (oligo-FapyG) with clustered DNA lesions (CDLs) containing both the above types of damage at the M06-2x/6-31++G** level of theory in the condensed phase. Furthermore, the electronic properties of oligo-FapyG were analysed in both equilibrated and non-equilibrated solvation-solute interaction modes. The vertical/adiabatic ionization potential (VIP, AIP) and electron affinity (VEA, AEA) of the investigated ds-oligo were found as follows in [eV]: 5.87/5.39 and -1.41/-2.09, respectively. The optimization of the four ds-DNA spatial geometries revealed that the transFapydG was energetically privileged. Additionally, CDLs were found to have little influence on the ds-oligo structure. Furthermore, for the FapyGC base-pair isolated from the discussed ds-oligo, the ionization potential and electron affinity values were higher than those assigned to OXOGC. Finally, a comparison of the influence of FapyGC and OXOGC on charge transfer revealed that, in contrast to the OXOGC base-pair, which, as expected, acted as a radical cation/anion sink in the oligo-FapyG structure, FapyGC did not significantly affect charge transfer (electron-hole and excess-electron). The results presented below indicate that 7,8-dihydro-8-oxo-2'-deoxyguanosine plays a significant role in charge transfer through ds-DNA containing CDL and indirectly has an influence on the DNA lesion recognition and repair process. In contrast, the electronic properties obtained for 2,6-diamino-4-hydroxy-5-foramido-2'deoxypyrimidine were found to be too weak to compete with OXOG to influence charge transfer through the discussed ds-DNA containing CDL. Because increases in multi-damage site formation are observed during radio- or chemotherapy, understanding their role in the above processes can be crucial for the efficiency and safety of medical cancer treatment.

Nucleotide excision repair: a versatile and smart toolkit.

Nucleotide excision repair (NER) is a major pathway to deal with bulky adducts induced by various environmental toxins in all cellular organisms. The two sub-pathways of NER, global genome repair (GGR) and transcription-coupled repair (TCR), differ in the damage recognition modes. In this review, we describe the molecular mechanism of NER in mammalian cells, especially the details of damage recognition steps in both sub-pathways. We also introduce new sequencing methods for genome-wide mapping of NER, as well as recent advances about NER in chromatin by these methods. Finally, the roles of NER factors in repairing oxidative damages and resolving R-loops are discussed.

"Flexible hinge" dynamics in mismatched DNA revealed by fluorescence correlation spectroscopy.

Altered unwinding/bending fluctuations at DNA lesion sites are implicated as plausible mechanisms for damage sensing by DNA-repair proteins. These dynamics are expected to occur on similar timescales as one-dimensional (1D) diffusion of proteins on DNA if effective in stalling these proteins as they scan DNA. We examined the flexibility and dynamics of DNA oligomers containing 3 base pair (bp) mismatched sites specifically recognized in vitro by nucleotide excision repair protein Rad4 (yeast ortholog of mammalian XPC). A previous Forster resonance energy transfer (FRET) study mapped DNA conformational distributions with cytosine analog FRET pair primarily sensitive to DNA twisting/unwinding deformations (Chakraborty et al. Nucleic Acids Res. 46: 1240-1255 (2018)). These studies revealed B-DNA conformations for nonspecific (matched) constructs but significant unwinding for mismatched constructs specifically recognized by Rad4, even in the absence of Rad4. The timescales of these unwinding fluctuations, however, remained elusive. Here, we labeled DNA with Atto550/Atto647N FRET dyes suitable for fluorescence correlation spectroscopy (FCS). With these probes, we detected higher FRET in specific, mismatched DNA compared with matched DNA, reaffirming unwinding/bending deformations in mismatched DNA. FCS unveiled the dynamics of these spontaneous deformations at ~ 300 µs with no fluctuations detected for matched DNA within the ~ 600 ns-10 ms FCS time window. These studies are the first to visualize anomalous unwinding/bending fluctuations in mismatched DNA on timescales that overlap with the < 500 µs "stepping" times of repair proteins on DNA. Such "flexible hinge" dynamics at lesion sites could arrest a diffusing protein to facilitate damage interrogation and recognition.

A Peek Inside the Machines of Bacterial Nucleotide Excision Repair.

Double stranded DNA (dsDNA), the repository of genetic information in bacteria, archaea and eukaryotes, exhibits a surprising instability in the intracellular environment; this fragility is exacerbated by exogenous agents, such as ultraviolet radiation. To protect themselves against the severe consequences of DNA damage, cells have evolved at least six distinct DNA repair pathways. Here, we review recent key findings of studies aimed at understanding one of these pathways: bacterial nucleotide excision repair (NER). This pathway operates in two modes: a global genome repair (GGR) pathway and a pathway that closely interfaces with transcription by RNA polymerase called transcription-coupled repair (TCR). Below, we discuss the architecture of key proteins in bacterial NER and recent biochemical, structural and single-molecule studies that shed light on the lesion recognition steps of both the GGR and the TCR sub-pathways. Although a great deal has been learned about both of these sub-pathways, several important questions, including damage discrimination, roles of ATP and the orchestration of protein binding and conformation switching, remain to be addressed.

Kinetic Milestones of Damage Recognition by DNA Glycosylases of the Helix-Hairpin-Helix Structural Superfamily.

X-ray data show that DNA glycosylases, which initiate the pathway of base excision repair in DNA, belong to six structural superfamilies. Here, we provide an overview of the latest results of kinetic studies on the mechanisms of specific recognition of a damaged nucleotide at the early steps of DNA repair by human (OGG1 and MBD4) or Escherichia coli (Nth and MutY) N-DNA-glycosylases belonging to superfamily Helix-hairpin-Helix (HhH). A comparison of real-time conformational transformations of DNA glycosylases and DNA with the structural data obtained for free enzymes and their complexes with substrates and intermediates have made it possible to build molecular-kinetic models of the enzymatic processes. These models have allowed researchers to associate the conformational transitions of the interacting molecules with elementary steps of an enzymatic process. Additionally, these models have revealed the stages that make the largest contribution to the specificity of the enzyme for DNA substrates. These data provide an opportunity to gain further insight into the structural and dynamic principles underlying the enzymatic processes that ensure highly efficient functioning of the repair-protective system of all living organisms and that maintain DNA integrity.

Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex.

DNA damage repair starts with the recognition of damaged sites from predominantly normal DNA. In eukaryotes, diverse DNA lesions from environmental sources are recognized by the xeroderma pigmentosum C (XPC) nucleotide excision repair complex. Studies of Rad4 (radiation-sensitive 4; yeast XPC ortholog) showed that Rad4 "opens" up damaged DNA by inserting a ß-hairpin into the duplex and flipping out two damage-containing nucleotide pairs. However, this DNA lesion "opening" is slow (˜5-10 ms) compared with typical submillisecond residence times per base pair site reported for various DNA-binding proteins during 1D diffusion on DNA. To address the mystery as to how Rad4 pauses to recognize lesions during diffusional search, we examine conformational dynamics along the lesion recognition trajectory using temperature-jump spectroscopy. Besides identifying the ˜10-ms step as the rate-limiting bottleneck towards opening specific DNA site, we uncover an earlier ˜100- to 500-µs step that we assign to nonspecific deformation (unwinding/"twisting") of DNA by Rad4. The ß-hairpin is not required to unwind or to overcome the bottleneck but is essential for full nucleotide-flipping. We propose that Rad4 recognizes lesions in a step-wise "twist-open" mechanism, in which preliminary twisting represents Rad4 interconverting between search and interrogation modes. Through such conformational switches compatible with rapid diffusion on DNA, Rad4 may stall preferentially at a lesion site, offering time to open DNA. This study represents the first direct observation, to our knowledge, of dynamical DNA distortions during search/interrogation beyond base pair breathing. Submillisecond interrogation with preferential stalling at cognate sites may be common to various DNA-binding proteins.

Molecular mechanisms and genomic maps of DNA excision repair in Escherichia coli and humans.

Nucleotide excision repair is a major DNA repair mechanism in all cellular organisms. In this repair system, the DNA damage is removed by concerted dual incisions bracketing the damage and at a precise distance from the damage. Here, we review the basic mechanisms of excision repair in Escherichia coli and humans and the recent genome-wide mapping of DNA damage and repair in these organisms at single-nucleotide resolution.

Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates.

How human DNA repair proteins survey the genome for UV-induced photoproducts remains a poorly understood aspect of the initial damage recognition step in nucleotide excision repair (NER). To understand this process, we performed single-molecule experiments, which revealed that the human UV-damaged DNA-binding protein (UV-DDB) performs a 3D search mechanism and displays a remarkable heterogeneity in the kinetics of damage recognition. Our results indicate that UV-DDB examines sites on DNA in discrete steps before forming long-lived, nonmotile UV-DDB dimers (DDB1-DDB2)2 at sites of damage. Analysis of the rates of dissociation for the transient binding molecules on both undamaged and damaged DNA show multiple dwell times over three orders of magnitude: 0.3-0.8, 8.1, and 113-126 s. These intermediate states are believed to represent discrete UV-DDB conformers on the trajectory to stable damage detection. DNA damage promoted the formation of highly stable dimers lasting for at least 15 min. The xeroderma pigmentosum group E (XP-E) causing K244E mutant of DDB2 found in patient XP82TO, supported UV-DDB dimerization but was found to slide on DNA and failed to stably engage lesions. These findings provide molecular insight into the loss of damage discrimination observed in this XP-E patient. This study proposes that UV-DDB recognizes lesions via multiple kinetic intermediates, through a conformational proofreading mechanism.

Ubiquitin at work: the ubiquitous regulation of the damage recognition step of NER.

Nucleotide excision repair (NER) removes a wide variety of helix distorting DNA lesions. NER comprises two damage recognition sub-pathways: GG-NER operates genome wide, whereas TC-NER specifically removes transcription-blocking lesions from the transcribed strand of actively transcribed genes. NER is a multistep process, which requires the concerted action of 30 proteins that need to be tightly controlled at the right time and place for efficient repair. Post-translational protein modifications (PTMs) are common regulators of complex protein networks. Several NER factors were shown to be modified by ubiquitin, whereas others are actively involved in the ubiquitin-proteasome system itself. PTMs by ubiquitylation can be swiftly induced in a reversible manner and have the ability to regulate protein function, localization or stability. This makes the regulation by ubiquitin highly suitable for the coordination of the complex NER reaction. Accumulating evidence, including proteome wide quantitative proteomics approaches, showed that especially NER factors involved in the damage recognition are regulated by ubiquitin, emphasizing the high level of regulation during the initiation of the NER reaction. In this review we will therefore focus on the different functions of ubiquitylation during the DNA damage recognition steps of NER.

Direct correlation of DNA binding and single protein domain motion via dual illumination fluorescence microscopy.

We report a dual illumination, single-molecule imaging strategy to dissect directly and in real-time the correlation between nanometer-scale domain motion of a DNA repair protein and its interaction with individual DNA substrates. The strategy was applied to XPD, an FeS cluster-containing DNA repair helicase. Conformational dynamics was assessed via FeS-mediated quenching of a fluorophore site-specifically incorporated into XPD. Simultaneously, binding of DNA molecules labeled with a spectrally distinct fluorophore was detected by colocalization of the DNA- and protein-derived signals. We show that XPD undergoes thermally driven conformational transitions that manifest in spatial separation of its two auxiliary domains. DNA binding does not strictly enforce a specific conformation. Interaction with a cognate DNA damage, however, stabilizes the compact conformation of XPD by increasing the weighted average lifetime of this state by 140% relative to an undamaged DNA. Our imaging strategy will be a valuable tool to study other FeS-containing nucleic acid processing enzymes.

Dynamics and energetics of PCBP1 binding to severely oxidized RNA.

Oxidatively generated lesions such as 8-oxo-7, 8-dihydroguanine (8-oxoG) on RNA strands constitute a hallmark marker of the oxidative stress in the cell. Poly-C binding protein 1 (PCBP1) is able to specifically recognize severely damaged RNA strands containing two 8-oxoG lesions separated by five nucleobases, which trigger a signaling pathway leading to cell apoptosis. We apply an in silico protocol based on microsecond timescale all-atom classical molecular dynamics simulations associated with conformational and energy analyses to unveil the specific recognition mechanism at a molecular level. By comparing the RNA and protein behavior for sequences with six different damage profiles, our results highlight an allosteric mechanism, allowing a stronger binding of the oxidized guanine at position 9 only if another 8-oxoG lesion is present at position 15, in full agreement with experiments. We assess the role of lysine K23 and the additional ketone group of the oxidized guanine, thanks to computational site-directed mutagenesis.

Impact of DNA sequences on DNA 'opening' by the Rad4/XPC nucleotide excision repair complex.

Rad4/XPC recognizes diverse DNA lesions to initiate nucleotide excision repair (NER). However, NER propensities among lesions vary widely and repair-resistant lesions are persistent and thus highly mutagenic. Rad4 recognizes repair-proficient lesions by unwinding ('opening') the damaged DNA site. Such 'opening' is also observed on a normal DNA sequence containing consecutive C/G's (CCC/GGG) when tethered to Rad4 to prevent protein diffusion. However, it was unknown if such tethering-facilitated DNA 'opening' could occur on any DNA or if certain structures/sequences would resist being 'opened'. Here, we report that DNA containing alternating C/G's (CGC/GCG) failed to be opened even when tethered; instead, Rad4 bound in a 180°-reversed manner, capping the DNA end. Fluorescence lifetime studies of DNA conformations in solution showed that CCC/GGG exhibits local pre-melting that is absent in CGC/GCG. In MD simulations, CGC/GCG failed to engage Rad4 to promote 'opening' contrary to CCC/GGG. Altogether, our study illustrates how local sequences can impact DNA recognition by Rad4/XPC and how certain DNA sites resist being 'opened' even with Rad4 held at that site indefinitely. The contrast between CCC/GGG and CGC/GCG sequences in Rad4-DNA recognition may help decipher a lesion's mutagenicity in various genomic sequence contexts to explain lesion-determined mutational hot and cold spots.

Light-induced modulation of DNA recognition by the Rad4/XPC damage sensor protein.

Biomolecular structural changes upon binding/unbinding are key to their functions. However, characterization of such dynamical processes is difficult as it requires ways to rapidly and specifically trigger the assembly/disassembly as well as ways to monitor the resulting changes over time. Recently, various chemical strategies have been developed to use light to trigger changes in oligonucleotide structures, and thereby their activities. Here we report that photocleavable DNA can be used to modulate the DNA binding of the Rad4/XPC DNA repair complex using light. Rad4/XPC specifically recognizes diverse helix-destabilizing/distorting lesions including bulky organic adduct lesions and functions as a key initiator for the eukaryotic nucleotide excision repair (NER) pathway. We show that the 6-nitropiperonyloxymethyl (NPOM)-modified DNA is recognized by the Rad4 protein as a specific substrate and that the specific binding can be abolished by light-induced cleavage of the NPOM group from DNA in a dose-dependent manner. Fluorescence lifetime-based analyses of the DNA conformations suggest that free NPOM-DNA retains B-DNA-like conformations despite its bulky NPOM adduct, but Rad4-binding causes it to be heterogeneously distorted. Subsequent extensive conformational searches and molecular dynamics simulations demonstrate that NPOM in DNA can be housed in the major groove of the DNA, with stacking interactions among the nucleotide pairs remaining largely unperturbed and thus retaining overall B-DNA conformation. Our work suggests that photoactivable DNA may be used as a DNA lesion surrogate to study DNA repair mechanisms such as nucleotide excision repair.

Automatic Concrete Damage Recognition Using Multi-Level Attention Convolutional Neural Network.

There has been an increase in the deterioration of buildings and infrastructure in dense urban regions, and several defects in the structures are being exposed. To ensure the effective diagnosis of building conditions, vision-based automatic damage recognition techniques have been developed. However, conventional image processing techniques have some limitations in real-world situations owing to their manual feature extraction approach. To overcome these limitations, a convolutional neural network-based image recognition technique was adopted in this study, and a convolution-based concrete multi-damage recognition neural network (CMDnet) was developed. The image datasets consisted of 1981 types of concrete surface damages, including surface cracks, rebar exposure and delamination, as well as intact. Furthermore, it was experimentally demonstrated that the proposed model could accurately classify the damage types. The results obtained in this study reveal that the proposed model can recognize the different damage types from digital images of the surfaces of concrete structures. The trained CMDnet demonstrated a damage-detection accuracy of 98.9%. Moreover, the proposed model could be applied in automatic damage detection networks to achieve superior performance with regard to concrete surface damage detection and recognition, as well as accelerating efficient damage identification during the diagnosis of deteriorating structures used in civil engineering applications.

Mechanism and regulation of DNA damage recognition in nucleotide excision repair.

Nucleotide excision repair (NER) is a versatile DNA repair pathway, which can remove an extremely broad range of base lesions from the genome. In mammalian global genomic NER, the XPC protein complex initiates the repair reaction by recognizing sites of DNA damage, and this depends on detection of disrupted/destabilized base pairs within the DNA duplex. A model has been proposed that XPC first interacts with unpaired bases and then the XPD ATPase/helicase in concert with XPA verifies the presence of a relevant lesion by scanning a DNA strand in 5'-3' direction. Such multi-step strategy for damage recognition would contribute to achieve both versatility and accuracy of the NER system at substantially high levels. In addition, recognition of ultraviolet light (UV)-induced DNA photolesions is facilitated by the UV-damaged DNA-binding protein complex (UV-DDB), which not only promotes recruitment of XPC to the damage sites, but also may contribute to remodeling of chromatin structures such that the DNA lesions gain access to XPC and the following repair proteins. Even in the absence of UV-DDB, however, certain types of histone modifications and/or chromatin remodeling could occur, which eventually enable XPC to find sites with DNA lesions. Exploration of novel factors involved in regulation of the DNA damage recognition process is now ongoing.

Mechanism and regulation of DNA damage recognition in mammalian nucleotide excision repair.

Nucleotide excision repair (NER) is a versatile DNA repair pathway that eliminates various helix-distorting base lesions such as ultraviolet (UV)-induced photolesions. Several recessive human disorders, such as xeroderma pigmentosum (XP), are caused by hereditary defects in NER, implying that the pathway plays critical roles in suppressing diverse pathogenic processes, including carcinogenesis. In general, discrimination of lesion sites from intact DNA, which is present in vast excess, is a key determinant of the overall efficiency of DNA repair. In mammalian cells, global genomic NER lesion recognition is initiated by the XPC protein complex, which achieves broad DNA-binding specificity by sensing destabilized base pairs rather than lesions per se. To avert unnecessary incisions at lesion-free sites, and thereby ensure the fidelity of the repair system, transcription factor IIH and the XPA protein then verify the presence of relevant lesions at suspicious sites bound by XPC. In the case of UV-induced photolesions, a specialized lesion sensor called UV-damaged DNA-binding protein (UV-DDB) contributes to efficient lesion recognition and the recruitment of XPC to lesion sites. The ubiquitin-proteasome system plays a crucial role in the handoff of lesions from UV-DDB to XPC and the subsequent NER process. In addition, recognition of lesions targeted by global genomic NER is intricately regulated by higher-order chromatin structures, which play distinct roles depending on the type of lesion.

Ethanol-induced DNA repair in neural stem cells is transforming growth factor ß1-dependent.

Following neurotoxic damage, cells repair their DNA, and survive or undergo apoptosis. This study tests the hypothesis that ethanol induces a DNA damage response (DDR) in neural stem cells (NSCs) that promotes excision repair (ER) and this repair is influenced by the growth factor environment. Non-immortalized NSCs treated with fibroblast growth factor 2 or transforming growth factor (TGF) ß1 were exposed to ethanol. Ethanol increased total DNA damage, reactive oxygen species, and oxidized DNA bases. TGFß1 potentiated these toxic effects. Transcriptional analyses of cultured NSCs revealed ethanol-induced increases in transcripts related to the DDR (e.g., Hus1 and p53), base ER (e.g., Mutyh and Nthl1), and nucleotide ER (e.g., Xpc), particularly in the presence of TGFß1. Expression and activity of ER proteins were affected by ethanol. Similar changes occurred in proliferating cells of ethanol-treated mouse fetuses. Ethanol-induced DNA repair in NSCs depends on the ambient growth factors. Gene products for DNA repair in stem cells are among the first biomarkers identifying fetal alcohol-induced damage.