DNA Length Research Articles

First report of papaya leaf curl virus and its associated papaya leaf curl betasatellite infecting Catharanthus roseus plants in India

ABSTRACT Catharanthus roseus (Madagascar periwinkle) plants exhibiting typical begomovirus symptoms, i.e., leaf yellowing and curling were found in Lakshmangarh, Rajasthan, India. Symptomatic and healthy leaf samples were collected and subjected to DNA extraction and polymerase chain reaction, using universal primers conserved for the coat protein region of begomoviruses. The full length of viral DNA was amplified by rolling circular amplification (RCA) followed by cloning and sequencing. We identified the viral DNA sequence as papaya leaf curl virus (PaLCuV: KX353619) and papaya leaf curl betasatellite (PaLCuB: KX353621). Analysis of complete viral DNA component revealed the highest similarity (93%) with Papaya leaf curl virus, while sequence data of betasatellite showed maximum (93%) identity with Papaya leaf curl betasatellite. To our knowledge, this is the first account of these two types of geminivirus DNA molecules harbouring together in C. roseus plants.

Dynamic DNA Shortening by Telomere-Binding Protein Cdc13.

Telomeres are essential for chromosome maintenance. Cdc13 is a single-stranded telomeric DNA binding protein that caps telomeres and regulates telomerase function in yeast. Although specific binding of Cdc13 to telomeric DNA is critical for telomere protection, the detail mechanism how Cdc13-DNA complex protects telomere is unclear. Using two single-molecule methods, tethered particle motion and atomic force microscopy, we demonstrate that specific binding of Cdc13 on single-stranded telomeric DNA shortens duplex DNA into distinct states differed by ∼70-80 base pairs. DNA shortening by Cdc13 is dynamic and independent of duplex DNA sequences or length. Significantly, we found that Pif1 helicase is incapable of removing Cdc13 from the shortened DNA-Cdc13 complex, suggesting that Cdc13 forms structurally stable complex by shortening of the bound DNA. Together our data identified shortening of DNA by Cdc13 and provided an indication for efficient protection of telomere ends by the shortened DNA-Cdc13 complex.

Label-free highly sensitive detection of DNA approximate length and concentration by impedimetric CRISPR-dCas9 based biosensor technology

In this study, we designed a CRISPR-dCas9-based biosensor with potential clinical use in glioblastoma subtype discrimination through detection of isocitrate dehydrogenase R132H (IDH) mutation status. The electrode was modified to detect mutant DNA cysteamine (Cys), PAMAM, dCas9 and sgRNA for R132H mutations, respectively. The biosensor system we proposed was able not only to detect mutant DNA, but also to measure the approximate length of DNA. Therefore, it can be considered that the biosensor technology that we developed is novel in the field of DNA biosensors. Another superior capability of the biosensor system is that it can simultaneously measure DNA concentration by electrochemical impedance spectroscopy and DNA length by capacitive detection, which lowers the concentration-based false-positive signals. The calibration range was obtained between 100 fM and 1000 fM, LOD and LOQ were also calculated as 33.96 fM and 102.91 fM respectively. Moreover, thanks to the sensitivity of the capacitive detection, the biosensor was able to discriminate the same EIS signals of the 200 bp and 250 fM concentration data and 1000 bp and 50 fM concentration data. In conclusion, the biosensor was capable of detect target DNA and DNA length, simultaneously in minutes.

Effects of size, cooperativity, and competitive binding on protein positioning on DNA

Accurate positioning of proteins on chromosomal DNA is crucial for its proper organization as well as gene transcription regulation. Recent experiments revealed existence of periodic patterns of nucleoprotein complexes on DNA, which frequently cannot be explained by sequence-dependent binding of proteins. Previous theoretical studies suggest that such patterns typically emerge as a result of the proteins’ volume-exclusion effect. However, the role of other physical factors in patterns’ formation, such as the length of DNA, its sequence heterogeneity, and protein binding cooperativity/binding competition to DNA, remains unclear. To address these less understood yet important aspects, we investigated potential effects of these factors on protein positioning on finite-size DNA by using transfer-matrix calculations. It has been found that upon binding to DNA, proteins form oscillatory patterns that span over the length of up to ∼10 times the size of the protein binding site, with the shape of the patterns being strongly dependent on the length of DNA and the proteins’ binding cooperativity to DNA. Furthermore, calculations showed that small variations in the proteins’ affinity to DNA due to its sequence heterogeneity do not much change the main geometric characteristics of the observed protein patterns. Finally, competition between two different types of proteins for binding to DNA has been found to lead to formation of highly diverse and complex alternating positioning of the two proteins. Altogether, these results provide new insights into the roles of physicochemical properties of proteins, the DNA length, and DNA-binding competition between proteins in formation of protein positioning patterns on DNA.

Determining Trap Compliances, Microsphere Size Variations, and Response Linearities in Single DNA Molecule Elasticity Measurements with Optical Tweezers.

We previously introduced the use of DNA molecules for calibration of biophysical force and displacement measurements with optical tweezers. Force and length scale factors can be determined from measurements of DNA stretching. Trap compliance can be determined by fitting the data to a nonlinear DNA elasticity model, however, noise/drift/offsets in the measurement can affect the reliability of this determination. Here we demonstrate a more robust method that uses a linear approximation for DNA elasticity applied to high force range (25–45 pN) data. We show that this method can be used to assess how small variations in microsphere sizes affect DNA length measurements and demonstrate methods for correcting for these errors. We further show that these measurements can be used to check assumed linearities of system responses. Finally, we demonstrate methods combining microsphere imaging and DNA stretching to check the compliance and positioning of individual traps.

DNA length tunes the fluidity of DNA-based condensates

Living organisms typically store their genomic DNA in a condensed form. Mechanistically, DNA condensation can be driven by macromolecular crowding, multivalent cations, or positively charged proteins. At low DNA concentration, condensation triggers the conformational change of individual DNA molecules into a compacted state, with distinct morphologies. Above a critical DNA concentration, condensation goes along with phase separation into a DNA-dilute and a DNA-dense phase. The latter DNA-dense phase can have different material properties and has been reported to be rather liquid-like or solid-like depending on the characteristics of the DNA and the solvent composition. Here, we systematically assess the influence of DNA length on the properties of the resulting condensates. We show that short DNA molecules with sizes below 1 kb can form dynamic liquid-like assemblies when condensation is triggered by polyethylene glycol and magnesium ions, binding of linker histone H1, or nucleosome reconstitution in combination with linker histone H1. With increasing DNA length, molecules preferentially condense into less dynamic more solid-like assemblies, with phage λ-DNA with 48.5 kb forming mostly solid-like assemblies under the conditions assessed here. The transition from liquid-like to solid-like condensates appears to be gradual, with DNA molecules of roughly 1–10 kb forming condensates with intermediate properties. Titration experiments with linker histone H1 suggest that the fluidity of condensates depends on the net number of attractive interactions established by each DNA molecule. We conclude that DNA molecules that are much shorter than a typical human gene are able to undergo liquid-liquid phase separation, whereas longer DNA molecules phase separate by default into rather solid-like condensates. We speculate that the local distribution of condensing factors can modulate the effective length of chromosomal domains in the cell. We anticipate that the link between DNA length and fluidity established here will improve our understanding of biomolecular condensates involving DNA.

Synthesis and Characterization of Click Nucleic Acid Conjugated Polymeric Microparticles for DNA Delivery Applications.

Microparticle-mediated nucleic acid delivery is a popular strategy to achieve therapeutic outcomes via antisense gene therapy. However, current methods used to fabricate polymeric microparticles suffer from suboptimal properties such as particle polydispersity and low encapsulation efficiency. Here, a new particulate delivery system based on step-growth thiol-Michael dispersion polymerization is reported in which a low polydispersity microparticle is functionalized with a synthetic nucleic acid mimic, namely, click nucleic acids (CNA). CNA oligomers, exhibiting an average length of approximately four nucleic acid repeat units per chain for both adenine and thymine bases, were successfully conjugated to excess thiols present in the microparticles. Effective DNA loading was obtained by simple mixing, and up to 6 ± 2 pmol of complementary DNA/mg of particle was achieved, depending on the length of DNA used. In addition, DNA loading was orders of magnitude less for noncomplementary sequences and sequences containing an alternating base mismatch. The DNA release properties were evaluated, and it was found that release could be triggered by sudden changes in temperature but was unaffected over a range of pH. Finally, phagocytosis of loaded microparticles was observed by confocal microscopy and corroborated by an increase in cellular metabolic activity up to 90%. Overall, this work suggests that CNA functionalized microparticles could be a promising platform for controlled DNA delivery.

Characterization of human serum albumin modified by hair dye component, 4-chloro-1,2-phenylenediamine: Role in protein aggregation, redox biology and cytotoxicity

Hair dyes and their components have been described as mutagenic and carcinogenic. They penetrate the skin, undergo auto-oxidation, yield protein reactive derivatives and modify proteins in an irreversible manner. Investigations into the impact of aromatic diamines on protein structures are expected to have a significant clinical value. This study documents the strong potential of 4-chloro-1, 2-phenylenediamine (4-Cl-o-PDA), a hair dye component, in the modification of human serum albumin (HSA). HSA was modified in vitro by 4-Cl-o-PDA and changes in tertiary and secondary structural characteristics were analyzed by UV–visible, fluorescence and far UV circular dichroism, fourier transform infrared spectroscopical techniques and ANS binding assay. Interaction of 4-Cl-o-PDA and HSA was studied by molecular docking and drug displacement assay. Formation of aggregates and inhibitory effect of antioxidant curcumin was analyzed by ThT binding assay, ThT fluorescence and electron microscopy. Changes in oxidation status of HSA were studied by thiol estimation and NBT assay. Effect of 4-Cl-o-PDA on cellular redox status, DNA integrity and cytotoxicity was analyzed in lymphocytes using dichlorofluorescein method, comet assay and MTT assay. We observed gross structural damage in HSA modified by 4-Cl-o-PDA. Modification led to unmasking of hydrophobic clusters, loss of its alpha helix and an increase in beta strand and unordered structure in HSA. 4-Cl-o-PDA strongly binds at sub-domain IB of HSA. HSA modified by 4-Cl-o-PDA formed amyloid fibrillar aggregates, whose formation was inhibited by antioxidant treatment. Modified HSA showed reduction of free sulfydryl content. 4-Cl-o-PDA-HSA led to the generation of reactive oxygen species in human lymphocytes. Modified HSA caused an increase in the tail length of DNA indicating enhanced single strand DNA breaks in cells. Modified protein was found to be genotoxic and cytotoxic. This works offers insights in the role of hair dye components in the biological world.

Assessment of Antioxidant Effect of Beta-Glucan on the Whole Blood Oxidative DNA Damage with the Comet Assay in Colorectal Cancer.

The present study aims to evaluate the antioxidant effect of beta-glucan on oxidative DNA damage by comet assay. A total of 19 adult females and males diagnosed with stage 3-4 colorectal cancer and a control group of 20 age-matched healthy subjects were enrolled in the study. Blood samples of the participants were analyzed using Comet Assay for the parameters of DNA damage. Significantly increased DNA damage was observed in patients versus the control group as indicated by greater values of tail moment, tail percent DNA and tail length. Following incubation with β-glucan, a substantial reduction was found in the aforementioned parameters of DNA damage. Comet assay revealed significant levels of endogenous DNA damage in patients as shown by remarkable increases in the tail moment, the percentage of DNA in the tail and the tail length values, in comparison with the control group. Following treatment of fresh whole blood with β-glucan incubation, DNA damages were significantly reduced, but lower values were observed after β-glucan incubation in the patient group versus control group. β-Glucan was found to reduce DNA damage substantially in colorectal cancer patients and show antimutagenic effects. Our results suggested that dietary β-glucan intake might be important in the genesis of colorectal cancer tumors.

One-dimensional diffusion of TrpR along DNA enhances its affinity for the operator by chemical ratchet mechanism

Several DNA-binding proteins show the affinities for their specific DNA sites that positively depend on the length of DNA harboring the sites, i. e. antenna effect. DNA looping can cause the effect for proteins with two or more DNA binding sites, i. e. the looping mechanism. One-dimensional diffusion also has been suggested to cause the effect for proteins with single DNA sites, the diffusion mechanism, which could violate detailed balance. We addressed which mechanism is possible for E. coli TrpR showing 104-fold antenna effect with a single DNA binding site. When a trpO-harboring DNA fragment was connected to a nonspecific DNA with biotin-avidin connection, the otherwise sevenfold antenna effect disappeared. This result denies the looping mechanism with an unknown second DNA binding site. The 3.5-fold repression by TrpR in vivo disappeared when a tight LexA binding site was introduced at various sites near the trpO, suggesting that the binding of LexA blocks one-dimensional diffusion causing the antenna effect. These results are consistent with the chemical ratchet recently proposed for TrpR-trpO binding to solve the deviation from detailed balance, and evidence that the antenna effect due to one-dimensional diffusion exists in cells.

Modeling Effects of Surface Properties and Probe Density for Nanoscale Biosensor Design: A Case Study of DNA Hybridization near Surfaces.

Electrochemical biosensors have extremely robust applications while offering ease of preparation, miniaturization, and tunability. By adjusting the arrangement and properties of immobilized probes on the sensor surface to optimize target-probe association, one can design highly sensitive and efficient sensors. In electrochemical nucleic acid biosensors, a self-assembled monolayer (SAM) is widely used as a tunable surface with inserted DNA or RNA probes to detect target sequences. The effects of inhomogeneous probe distribution across surfaces are difficult to study experimentally due to inadequate resolution. Regions of high probe density may inhibit hybridization with targets, and the magnitude of the effect may vary depending on the hybridization mechanism on a given surface. Another fundamental question concerns diffusion and hybridization of DNA taking place on surfaces and whether it speeds up or hinders molecular recognition. We used all-atom Brownian dynamics simulations to help answer these questions by simulating the hybridization process of single-stranded DNA (ssDNA) targets with a ssDNA probe on polar, nonpolar, and anionic SAMs at three different probe surface densities. Moreover, we simulated three tightly packed probe clusters by modeling clusters with different interprobe spacing on two different surfaces. Our results indicate that hybridization efficiency depends strongly on finding a balance that allows attractive forces to steer target DNA toward probes without anchoring it to the surface. Furthermore, we found that the hybridization rate becomes severely hindered when interprobe spacing is less than or equal to the target DNA length, proving the need for a careful design to both enhance target-probe association and avoid steric hindrance. We developed a general kinetic model to predict hybridization times and found that it works accurately for typical probe densities. These findings elucidate basic features of nanoscale biosensors, which can aid in rational design efforts and help explain trends in experimental hybridization rates at different probe densities.

A critical role for linker DNA in higher-order folding of chromatin fibers.

Nucleosome-nucleosome interactions drive the folding of nucleosomal arrays into dense chromatin fibers. A better physical account of the folding of chromatin fibers is necessary to understand the role of chromatin in regulating DNA transactions. Here, we studied the unfolding pathway of regular chromatin fibers as a function of single base pair increments in linker length, using both rigid base-pair Monte Carlo simulations and single-molecule force spectroscopy. Both computational and experimental results reveal a periodic variation of the folding energies due to the limited flexibility of the linker DNA. We show that twist is more restrictive for nucleosome stacking than bend, and find the most stable stacking interactions for linker lengths of multiples of 10 bp. We analyzed nucleosomes stacking in both 1- and 2-start topologies and show that stacking preferences are determined by the length of the linker DNA. Moreover, we present evidence that the sequence of the linker DNA also modulates nucleosome stacking and that the effect of the deletion of the H4 tail depends on the linker length. Importantly, these results imply that nucleosome positioning in vivo not only affects the phasing of nucleosomes relative to DNA but also directs the higher-order structure of chromatin.

Nitrogen-doped carbon dots aid in the separation of ssDNA molecules of different length by capillary transient isotachophoresis (ctITP) with laser-induced fluorescence (LIF) detection

This study demonstrates a novel application of nitrogen-doped carbon dots (NCDs) to enable the separation of different lengths of single-stranded DNA (ssDNA) by eletrokinetic means. Carbon dots have recently found widespread application in the fields of sensing, diagnostics, and healthcare due to their biocompatibility and low toxicity. In light of growing interest in the use of ssDNA aptamers over antibodies in the fields of biosensor development and drug delivery, it is important to establish a simple and effective method for aptamer separation. In this study, we employed NCDs as buffer additives in a capillary electrophoresis (CE)-based method, giving rise to the separation of FAM-labeled ssDNA samples ranging from 32 to 100 bases in length, with resolutions ranging from 1.30 – 1.77. In particular, we adopted a capillary transient isotachophoresis (ctITP) system with laser-induced fluorescence (LIF) detection, with both the separation and sample buffers modified by the addition of 30 μg/mL NCDs. These nanomaterials were prepared by a simple hydrothermal method from a mixture of citric acid and ethylenediamine. The NCDs themselves are highly fluorescent and photostable. As components in the background electrolyte, they did not interfere with the fluorescence emission of the FAM-labeled DNA samples. Under the conditions employed, no separation could be achieved in the absence of the NCDs nor with undoped CDs. The results show that NCDs function as buffer additives capable of enhancing electrokinetic-based separations of ssDNA, and hence, provide a new application for these carbon nanomaterials.

DNA-Mediated Au-Au Dimer-Based Surface Plasmon Coupling Electrochemiluminescence Sensor for BRCA1 Gene Detection.

Herein, we constructed a DNA-mediated Au-Au dimer-based surface plasmon coupling electrochemiluminescence (SPC-ECL) sensor. In the SPC-ECL sensing system, graphite phase carbon nitride quantum dots (GCN QDs) worked as an ECL emitter. A DNA rigid chain structure was employed to connect two Au NPs in an equilateral triangle configuration to form the Au-Au dimers. Due to the hot spot effect, the designed Au-Au dimers had a strong electromagnetic field intensity, which can greatly enhance the ECL signal of GCN QDs than a single Au nanoparticle. The gap distance of dimers can be effectively regulated by the DNA length, which resulted in different electromagnetic field intensities. Therefore, the different SPC-ECL amplification effects on the GCN QD signal by Au-Au dimers have been revealed. The maximum ECL signal of GCN QDs can be enhanced fourfold based on the Au-Au dimers with a gap distance of 2 nm. Furthermore, the biosensor showed good analytical performance for the detection of breast cancer susceptibility gene 1 (BRCA1 genes) (1 fM-1 nM) with a detection limit of 0.83 fM. This work provided an effective and precise SPC-ECL sensing mode for the diagnosis and prognosis of breast cancer.

DNA Tracer Transport Through Porous Media—The Effect of DNA Length and Adsorption

AbstractArtificial tracer testing is an effective technique to identify fluid flow pathways and characterize subsurface hydrological properties. Synthetic DNA tracers, available in virtually unlimited number of unique variations, enable multiwell tracer testing and have the potential to improve the characterization of groundwater flowpaths enormously. This study investigated the effect of DNA length (i.e., number of base pairs) and adsorption on DNA tracer transport via well‐controlled laboratory experiments. Nine unique synthetic double‐stranded DNA tracers were designed with varying lengths (90–200 base pairs) and flowed with reference solute tracers (bromide and lithium) through columns packed with glass beads or Ottawa sand. The peak arrivals of DNA tracers were earlier than bromide in the glass‐bead column test, yet significantly retarded in the sand column test. In a given porous medium, DNA length did not have noticeable effects on peak arrival or tracer dispersion. However, DNA tracer recovery increased with increasing DNA length in the glass‐bead column test but decreased with increasing DNA length in the sand column test, likely resulting from the corresponding trends in partition coefficients as revealed by tracer transport modeling. Such reversed trends in different porous media likely resulted from the different likelihoods for longer DNA to have multisegment attachment during transport. Our findings suggest that both DNA length and the surface properties of the porous medium have notable effects on DNA tracer transport and hence need to be considered carefully in future tracer test designs in terms of site selection, DNA sequence design and data interpretation.

Oct4 Pioneers Genomic Nucleosomes in Motion

Gene regulation programs rely on dynamic changes in the structural packaging of genomic DNA. The DNA is stored in chromatin, a structure of arrays of nucleosomes with different degree of compaction and length of inter-nucleosomal linker DNA. In the nucleosome, 4 histone proteins form an octamer which wraps 147 basepairs of DNA around. Each histone is present twice and has a structured cores and intrinsically disordered terminal tails. Changes in chromatin structure are controlled by the DNA sequence, the conformational dynamics and post-translational modifications of histone tails, and by the binding of proteins to the nucleosomes.

DNA Folding by Multivalent Cations

Folding short lengths of DNA into loops is a fundamental area of research in the field of nanoengineering. While physical mechanisms of DNA folding may differ according to the multivalent cations involved in folding, the mechanical task is the same. Here, we compare the folding pathways and resulting loops for DNA folding by multivalent cations that are known to condense DNA. Specifically, we compared the looping of DNA by protamine (+21 and 5 kDA), hexaammine-cobalt (III) (+3 and 0.3 kDa), spermine (+4 and 0.2 kDa), and histone H1 (+42 and 20 kDA).

Single-Molecule Imaging Reveals the Mechanism Underlying Histone Loading of Schizosaccharomyces pombe AAA+ ATPase Abo1.

Chromatin dynamics is essential for maintaining genomic integrity and regulating gene expression. Conserved bromodomain-containing AAA+ ATPases play important roles in nucleosome organization as histone chaperones. Recently, the high-resolution cryo-electron microscopy structures of Schizosaccharomyces pombe Abo1 revealed that it forms a hexameric ring and undergoes a conformational change upon ATP hydrolysis. In addition, single-molecule imaging demonstrated that Abo1 loads H3-H4 histones onto DNA in an ATP hydrolysis-dependent manner. However, the molecular mechanism by which Abo1 loads histones remains unknown. Here, we investigated the details concerning Abo1-mediated histone loading onto DNA and the Abo1-DNA interaction using single-molecule imaging techniques and biochemical assays. We show that Abo1 does not load H2A-H2B histones. Interestingly, Abo1 deposits multiple copies of H3-H4 histones as the DNA length increases and requires at least 80 bp DNA. Unexpectedly, Abo1 weakly binds DNA regardless of ATP, and neither histone nor DNA stimulates the ATP hydrolysis activity of Abo1. Based on our results, we propose an allosteric communication model in which the ATP hydrolysis of Abo1 changes the configuration of histones to facilitate their deposition onto DNA.

Single-Particle Tracking of DNA-Binding Biomolecules in Cells: Power-Law Distributions of Dwell Times

Single-particle tracking (SPT) experiments have shown a truncated power-law distribution of dwell times for several DNA-binding species in the nucleus of living cells. Why this distribution? A standard explanation is Arrhenius escape from traps with exponentially distributed binding energies. But here the binding energies are roughly Gaussian, yielding roughly lognormal escape times. We therefore examine other mechanisms. The operational definition of SPT immobility is based on the dwell time within the volume of the effective SPT point spread function. The distribution of dwell times can be power-law at short times, but the exponent is constant and much less than observed. The dwell time problem has been treated in terms of revisits by a tracer to a trap, here recast as the number of revisits before the tracer leaves the volume of the effective SPT point spread function. The effect of revisits is limited because the sum of multiple escape times from a moderate trap is tame compared with single escapes from a deep trap, by the central limit theorem. Truncated power-law trapping yields transient anomalous subdiffusion, and obstructed diffusion includes two well-studied cases of this. In percolation the tracer concentration is low and the concentration of immobile obstacles is high. Anomalous subdiffusion is the result of static geometric trapping by dead ends, but how can these structures be made on the SPT length scale from DNA with a 50-nm persistence length? The other case is crowding, in which all species are mobile, the concentration is high, and the geometric traps are dynamic. We examine the effects of polydisperse crowders and of small concentrations of immobile species in a crowded system, to see whether crowding can bridge the gap between the SPT and DNA length scales.

An Autoinhibitory Role for the GRF Zinc Finger Domain of DNA Glycosylase NEIL3

DNA damage within a cell can cause damaging effects on genome stability if not properly corrected. Exogenous factors such as UV-light and environmental toxins, and endogenous factors including reactive metabolites and oxidative stress cause DNA damage in the form of modified DNA nucleobases. Base excision repair (BER) is the primary pathway to fix these lesions and begins with recognition and excision of the damaged nucleobase by DNA glycosylase enzymes. The DNA glycosylase, endonuclease VIII-like 3 (NEIL3), removes oxidized bases from single-stranded (ss) DNA, and unhooks interstrand cross-links (ICLs), which covalently tether opposing DNA strands and block transcription and replication. NEIL3 contains two C-terminal GRF-type zinc finger (Zf-GRFs) motifs absent in other NEIL paralogs and the role of the Zf-GRFs are unknown. Here we show the first crystal structure of the C-terminus of NEIL3 which includes a flexible head-to-tail configuration, predicted to bind ssDNA in many conformations. We show via electrophoretic mobility shift assays (EMSA) that the Zf-GRF domain binds with higher affinity than individual Zf-GRF motifs. Binding assays performed by fluorescence anisotropy showed DNA length dependence and DNA binding stoichiometry of the Zf-GRF domain. Functionally we show that the Zf-GRFs inhibit base excision activity of the NEIL3 glycosylase domain (NEIL3-GD) in trans and in cis. This autoinhibitory activity contrasts GRF-Zf domains of other DNA processing enzymes, which typically bind ssDNA to enhance catalytic activity, and suggests that the C-terminal of NEIL3 is involved in DNA damage recruitment and enzymatic regulation.