Mammalian N1-adenosine PARylation is a reversible DNA modification.

Poly-ADP-ribosylation (PARylation) is regarded as a protein-specific modification. However, some PARPs were recently shown to modify DNA termini in vitro. Here, we use ultrasensitive mass spectrometry (LC-MS/MS), anti-PAR antibodies, and anti-PAR reagents to show that mammalian DNA is physiologically PARylated and to different levels in primary tissues. Inhibition of PAR glycohydrolase (PARG) increases DNA PARylation, supporting that the modification is reversible. DNA PARylation requires PARP1 and in vitro PARP1 PARylates single-stranded DNA, while PARG reverts the modification. DNA PARylation occurs at the N1-position of adenosine residues to form N1-Poly(ADP-ribosyl)-deoxyadenosine. Through partial hydrolysis of mammalian gDNA we identify PAR-DNA via the diagnostic deamination product N1-ribosyl-deoxyinosine to occur in vivo. The discovery of N1-adenosine PARylation as a DNA modification establishes the conceptual and methodological framework to elucidate its biological relevance and extends the role of PARP enzymes.

The origin of genomic N6-methyl-deoxyadenosine in mammalian cells.

The proposal that N6-methyl-deoxyadenosine (m6dA) acts as an epigenetic mark in mammals remains controversial. Using isotopic labeling coupled to ultrasensitive mass spectrometry, we confirm the presence of low-level m6dA in mammalian DNA. However, the bulk of genomic m6dA originates from ribo-N6-methyladenosine, which is processed via the nucleotide-salvage pathway and misincorporated by DNA polymerases. Our results argue against m6dA acting as a heritable, epigenetic DNA mark in mammalian cells.

NEIL1 and NEIL2 DNA glycosylases protect neural crest development against mitochondrial oxidative stress.

Base excision repair (BER) functions not only in the maintenance of genomic integrity but also in active DNA demethylation and epigenetic gene regulation. This dual role raises the question if phenotypic abnormalities resulting from deficiency of BER factors are due to DNA damage or impaired DNA demethylation. Here we investigate the bifunctional DNA glycosylases/lyases NEIL1 and NEIL2, which act in repair of oxidative lesions and in epigenetic demethylation. Neil-deficiency in Xenopus embryos and differentiating mouse embryonic stem cells (mESCs) leads to a surprisingly restricted defect in cranial neural crest cell (cNCC) development. Neil-deficiency elicits an oxidative stress-induced TP53-dependent DNA damage response, which impairs early cNCC specification. Epistasis experiments with Tdg-deficient mESCs show no involvement of epigenetic DNA demethylation. Instead, Neil-deficiency results in oxidative damage specific to mitochondrial DNA, which triggers a TP53-mediated intrinsic apoptosis. Thus, NEIL1 and NEIL2 DNA glycosylases protect mitochondrial DNA against oxidative damage during neural crest differentiation.

GADD45 promotes locus-specific DNA demethylation and 2C cycling in embryonic stem cells.

Mouse embryonic stem cell (ESC) cultures contain a rare cell population of "2C-like" cells resembling two-cell embryos, the key stage of zygotic genome activation (ZGA). Little is known about positive regulators of the 2C-like state and two-cell stage embryos. Here we show that GADD45 (growth arrest and DNA damage 45) proteins, regulators of TET (TET methylcytosine dioxygenase)-mediated DNA demethylation, promote both states. Methylome analysis of Gadd45a,b,g triple-knockout (TKO) ESCs reveal locus-specific DNA hypermethylation of ∼7000 sites, which are enriched for enhancers and loci undergoing TET-TDG (thymine DNA glycosylase)-mediated demethylation. Gene expression is misregulated in TKOs, notably upon differentiation, and displays signatures of DNMT (DNA methyltransferase) and TET targets. TKOs manifest impaired transition into the 2C-like state and exhibit DNA hypermethylation and down-regulation of 2C-like state-specific genes. Gadd45a,b double-mutant mouse embryos display embryonic sublethality, deregulated ZGA gene expression, and developmental arrest. Our study reveals an unexpected role of GADD45 proteins in embryonic two-cell stage regulation.

Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation.

DNA 5-methylcytosine is a dynamic epigenetic mark with important roles in development and disease. In the Tet-Tdg demethylation pathway, methylated cytosine is iteratively oxidized by Tet dioxygenases, and unmodified cytosine is restored via thymine DNA glycosylase (Tdg). Here we show that human NEIL1 and NEIL2 DNA glycosylases coordinate abasic-site processing during TET-TDG DNA demethylation. NEIL1 and NEIL2 cooperate with TDG during base excision: TDG occupies the abasic site and is displaced by NEILs, which further process the baseless sugar, thereby stimulating TDG-substrate turnover. In early Xenopus embryos, Neil2 cooperates with Tdg in removing oxidized methylcytosines and specifying neural-crest development together with Tet3. Thus, Neils function as AP lyases in the coordinated AP-site handover during oxidative DNA demethylation.

GADD45a physically and functionally interacts with TET1.

DNA demethylation plays a central role during development and in adult physiology. Different mechanisms of active DNA demethylation have been established. For example, Growth Arrest and DNA Damage 45-(GADD45) and Ten-Eleven-Translocation (TET) proteins act in active DNA demethylation but their functional relationship is unresolved. Here we show that GADD45a physically interacts--and functionally cooperates with TET1 in methylcytosine (mC) processing. In reporter demethylation GADD45a requires endogenous TET1 and conversely TET1 requires GADD45a. On GADD45a target genes TET1 hyperinduces 5-hydroxymethylcytosine (hmC) in the presence of GADD45a, while 5-formyl-(fC) and 5-carboxylcytosine (caC) are reduced. Likewise, in global analysis GADD45a positively regulates TET1 mediated mC oxidation and enhances fC/caC removal. Our data suggest a dual function of GADD45a in oxidative DNA demethylation, to promote directly or indirectly TET1 activity and to enhance subsequent fC/caC removal.

DAZL regulates Tet1 translation in murine embryonic stem cells.

Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from a pristine naïve pluripotent state to a primed epiblast state. Addition of inhibitors of GSK3ß and MEK (so-called 2i conditions) pushes ESC cultures toward a more homogeneous naïve pluripotent state, but the molecular underpinnings of this naïve transition are not completely understood. Here, we demonstrate that DAZL, an RNA-binding protein known to play a key role in germ-cell development, marks a subpopulation of ESCs that is actively transitioning toward naïve pluripotency. Moreover, DAZL plays an essential role in the active reprogramming of cytosine methylation. We demonstrate that DAZL associates with mRNA of Tet1, a catalyst of 5-hydroxylation of methyl-cytosine, and enhances Tet1 mRNA translation. Overexpression of DAZL in heterogeneous ESC cultures results in elevated TET1 protein levels as well as increased global hydroxymethylation. Conversely, null mutation of Dazl severely stunts 2i-mediated TET1 induction and hydroxymethylation. Our results provide insight into the regulation of the acquisition of naïve pluripotency and demonstrate that DAZL enhances TET1-mediated cytosine hydroxymethylation in ESCs that are actively reprogramming to a pluripotent ground state.

Stable DNA aggregation by removal of counterions.

Negatively charged DNA can form extremely stable complexes with positively charged ions. These counterions are very difficult to remove from DNA; therefore, little is known about DNA behavior in their deficiency. We investigated whether removal of counterions from the strongly bound counterion layer would elicit any novel DNA properties or behaviors. In order to remove the tightly bound counterions, we used dialysis against deionized water in the presence of a strong (0.6 kV/cm) electric field. The electric field promoted the dissociation of the DNA-counterion complexes, while dialysis facilitated irreversible partitioning of counterions and DNA. Counterintuitively, when deprived of counterions, DNA precipitated from the solution into amorphous aggregates. The aggregates remained stable even when the electric field was turned off but readily redissolved when counterions were reintroduced. The phenomenon is likely explained by attraction of like-charged DNA polyions due to entropic-stabilization of condensed counterion layers.

Non-uniform velocity of homogeneous DNA in a uniform electric field: consequence of electric-field-induced slow dissociation of highly stable DNA-counterion complexes.

Identical molecules move with identical velocities when placed in a uniform electric field within a uniform electrolyte. Here we report that homogeneous DNA does not obey this fundamental rule. While most DNA moves with similar velocities, a fraction of DNA moves with velocities that vary within a multiple-fold range. The size of this irregular fraction increases several orders of magnitude when exogenous counterions are added to DNA. The irregular fraction decreases several orders of magnitude when DNA counterions are removed by dialysis against deionized water in the presence of a strong electric field (0.6 kV/cm). Dialysis without the field is ineffective in decreasing the size of irregular fraction. These results suggest that (i) DNA can form very stable complexes with counterions, (ii) these complexes can be dissociated by an electric field, and (iii) the observed non-uniform velocity of DNA is caused by electric-field-induced slow dissociation of these stable complexes. Our findings help to better understand a fundamental property of DNA: its interaction with counterions. In addition, these findings suggest a practical way of making electromigration of DNA more uniform: removal of strongly bound DNA counterions by electro-dialysis against deionized water.

DNA aptamers for as analytical tools for the quantitative analysis of DNA-dealkylating enzymes.

The AlkB family of oxygenases catalyze the removal of alkyl groups from nucleic acid substrates in an iron and 2-oxoglutarate-dependent manner and have roles including in DNA repair. To understand the biological functions of these DNA-dealkylating enzymes it is desirable to measure their expression levels in vitro and in vivo in complex biological matrixes. Quantitative analyses of the enzymes require affinity probes capable of binding AlkB family members selectively and with high affinity. Here we report that DNA aptamers can serve as efficient affinity probes for quantitative detection of such enzymes in vitro. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) was applied as a general tool for: (i) selection of DNA aptamers, (ii) characterization of binding parameters for the aptamers, and (iii) quantitative detection of the target in an aptamer-based affinity analysis. The selected aptamers have a range of K(d) values between 20 and 240nM. The aptamers enabled accurate quantitative analysis of AlkB even in the presence of the Escherichia coli cell lysate. Aptamers can likely be developed for other nucleic acid repair enzymes. They may also be developed for use in in vitro and potentially in vivo studies of known nucleic acid-modifying enzymes including for functional analysis.

Universal method for determining electrolyte temperatures in capillary electrophoresis.

Temperature increase in capillary electrophoresis (CE) due to Joule heating is an inherent limitation of this powerful separation technique. Active cooling systems can decrease the temperature of a large part of the capillary but they leave "hot spots" at the capillary ends which can completely ruin some CE analyses despite their short lengths. Here, we introduce a "universal method for determining electrolyte temperatures" (UMET) that can determine temperatures in both efficiently- and inefficiently-cooled parts of the capillary. UMET can be applied to all electrolytes, as it does not involve any probe; it requires only measuring current versus voltage for different voltages and processing the data using an iterative algorithm. To demonstrate the universality of UMET, we measured temperatures for electrolytes of different ionic strengths as well as for different capillary diameters. We further propose a "simplified universal method for predicting electrolyte temperatures" (SUMET) which only requires one measurement of current and voltage (that can be completed in 1 min) and uses two empirical equations to predict temperatures in the efficiently- and inefficiently-cooled parts of the capillary. The equations include several instrument-specific empirical parameters that are determined using a large set of current-voltage data obtained with UMET for a range of electrolytes and different capillaries. To demonstrate the utility of SUMET, we obtained the required data set for a Beckman MDQ CE instrument and produced all required empirical parameters that enable a user of this instrument to predict the temperature for every new experimental set in a matter of minutes. We confirmed the accuracy of SUMET by measuring the temperature-sensitive dissociation rate constant of a protein-DNA complex. We foresee that UMET will be used to produce instrument-specific empirical parameters for all CE instruments and then SUMET will be routinely used for temperature prediction in CE.

Electric field destabilizes noncovalent protein-DNA complexes.

Noncovalent protein-DNA interactions are involved in many vital biological processes. In cells, these interactions may take place in the environment of an electric field which originates from the plasma and organelle membranes and reaches strengths of 1 MV/cm. Moreover, protein-DNA interactions are often studied in vitro using an electric field as strong as 1 kV/cm, for example by electrophoresis. It is widely accepted that an electric field does not affect such interactions. Here we report on the first proof that an electric field of less than 1 kV/cm can destabilize the protein-DNA complexes through increasing the monomolecular rate constant of complex dissociation.

Noncooled capillary inlet: a source of systematic errors in capillary-electrophoresis-based affinity analyses.

Capillary electrophoresis (CE) serves as a platform for a large family of temperature-sensitive affinity methods. To control the electrolyte temperature, the heat generated during electrophoresis is removed by actively cooling the capillary. Short parts of the capillary, particularly at its inlet, are not actively cooled, however, and the electrolyte in this part is likely to be at an elevated temperature. Owing to their relatively short lengths, the noncooled parts have never been considered as a potential source of artifacts. Here we report for the first time that electrophoresis of the sample through the short noncooled capillary inlet can lead to large systematic errors in quantitative CE-based affinity analyses. Our findings suggest that the noncooled capillary inlet region, in spite of being short, is a source of significant artifacts that must be taken into consideration by developers and users of CE-based affinity methods. We propose a simple solution for this problem: moving the sample through the noncooled inlet into the cooled region by pressure or by a low-strength electric field to save it from exposure to the elevated temperature.

Temperature difference between the cooled and the noncooled parts of an electrolyte in capillary electrophoresis.

Joule heating always accompanies electrophoresis and unavoidably leads to a temperature increase of the electrolyte. The elevated temperatures are known to adversely affect the quality of separation and detection. To minimize the temperature increase in capillary electrophoresis (CE), Joule heat is removed by actively cooling the capillary. However, there are always small parts of the capillary, such as its inlet, outlet, and detection window, which are not actively cooled. The noncooled capillary inlet has been recently proven to have an elevated temperature which is high enough to significantly affect CE-based quantitative affinity analyses. The temperature difference between the cooled and noncooled regions has never been determined due to the lack of a suitable method. Here, we report on the first experimental determination of temperature in the cooled part of the capillary and the noncooled inlet region of the capillary. We found that, under typical CE conditions, with a low-conductivity run buffer, the temperature in the noncooled inlet exceeded the temperature in the cooled region by more than 15 °C. High-conductivity buffers are anticipated to have even greater temperature differences between the noncooled and cooled capillary parts. Our results strongly suggest the potential effect of the noncooled capillary regions on the quality of CE-based analyses, which cannot be ignored. The simplest way to avoid potential errors is to move the sample to the cooled region by pressure or by applying a low electric field.

Heat-associated field distortion in electro-migration techniques.

Electro-migration techniques, such as electrophoresis, are widely utilized in analytical sciences. If a single electrolyte is used, the field strength is typically assumed to be well-defined. Heat-associated field distortion (HAFD) has been suggested as a result of the nonuniform heat dissipation throughout the electrolyte; however, it has never been experimentally studied. Here, we experimentally demonstrated HAFD for the first time. We used capillary electrophoresis (CE) with a capillary having parts with different heat dissipation efficiencies. Our experiments showed a difference in field strength of approximately 1.5 times between the different parts of the capillary for a typical CE electrolyte. This result suggests that HAFD is a well pronounced phenomenon that can be a potential source of errors and instabilities in electro-migration experiments.

Direct analysis of enzyme-catalyzed DNA demethylation.

N/O-methylation of DNA can be cytotoxic and mutagenic; therefore, enzymes that reverse DNA methylation are essential for organism survival. Several 2-oxoglutarate-dependent oxygenases and methyltransferases that remove a methyl group from a methylated DNA base have been identified. Studies of their kinetics and search for their inhibitors have been retarded by the lack of an approach to directly quantitate DNA substrates and products that differ by a single methyl group. Here, we introduce such an approach, which is based on capillary electrophoresis with laser-induced fluorescence detection. We achieved baseline separation of a fluorescently labeled 15-nucleotide-long single-base methylated DNA substrate from its demethylated product, followed by its quantitative detection. We then used this approach to study the kinetics of AlkB-catalyzed DNA demethylation and screen a number of potential inhibitors of this reaction. Ten new inhibitors, which can be used as templates in developing therapies targeting AlkB-like enzymes, were identified. Our approach will be applicable for in vitro kinetic studies of known DNA demethylating and methylating enzymes and in the discovery of new ones.

Selection of aptamers for a protein target in cell lysate and their application to protein purification.

Functional genomics requires structural and functional studies of a large number of proteins. While the production of proteins through over-expression in cultured cells is a relatively routine procedure, the subsequent protein purification from the cell lysate often represents a significant challenge. The most direct way of protein purification from a cell lysate is affinity purification using an affinity probe to the target protein. It is extremely difficult to develop antibodies, classical affinity probes, for a protein in the cell lysate; their development requires a pure protein. Thus, isolating the protein from the cell lysate requires antibodies, while developing antibodies requires a pure protein. Here we resolve this loop problem. We introduce AptaPIC, Aptamer-facilitated Protein Isolation from Cells, a technology that integrates (i) the development of aptamers for a protein in cell lysate and (ii) the utilization of the developed aptamers for protein isolation from the cell lysate. Using MutS protein as a target, we demonstrate that this technology is applicable to the target protein being at an expression level as low as 0.8% of the total protein in the lysate. AptaPIC has the potential to considerably speed up the purification of proteins and, thus, accelerate their structural and functional studies.

Kinetic capillary electrophoresis-based affinity screening of aptamer clones.

DNA aptamers are single stranded DNA (ssDNA) molecules artificially selected from random-sequence DNA libraries for their specific binding to a certain target. DNA aptamers have a number of advantages over antibodies and promise to replace them in both diagnostic and therapeutic applications. The development of DNA aptamers involves three major stages: library enrichment, obtaining individual DNA clones, and the affinity screening of the clones. The purpose of the screening is to obtain the nucleotide sequences of aptamers and the binding parameters of their interaction with the target. Highly efficient approaches have been recently developed for the first two stages, while the third stage remained the rate-limiting one. Here, we introduce a new method for affinity screening of individual DNA aptamer clones. The proposed method amalgamates: (i) aptamer amplification by asymmetric PCR (PCR with a primer ratio different from unity), (ii) analysis of aptamer-target interaction, combining in-capillary mixing of reactants by transverse diffusion of laminar flow profiles (TDLFP) and affinity analysis using kinetic capillary electrophoresis (KCE), and (iii) sequencing of only aptamers with satisfying binding parameters. For the first time we showed that aptamer clones can be directly used in TDLFP/KCE-based affinity analysis without an additional purification step after asymmetric PCR amplification. We also demonstrated that mathematical modeling of TDLFP-based mixing allows for the determination of K(d) values for the in-capillary reaction of an aptamer and a target and that the obtained K(d) values can be used for the accurate affinity ranking of aptamers. The proposed method does not require the knowledge of aptamer sequences before screening, avoids lengthy (3-5 h) purification steps of aptamer clones, and minimizes reagent consumption to nanoliters.

Selection of smart small-molecule ligands: the proof of principle.

The development of drugs and diagnostics with desirable characteristics requires smart small-molecule ligandsligands with predefined binding parameters of interaction with the target. Here, we propose a general approach for selection of such ligands from highly diverse combinatorial libraries of small molecules by methods of kinetic capillary electrophoresis (KCE). We deduct three fundamental requirements for the combinatorial library to suit the KCE-based selection of smart ligands and suggest a universal design of the library for selecting smart small-molecule ligands: every small molecule in the library is tagged with DNA that encodes the structure of the molecule. Finally, we use several DNA-tagged small molecules, which represent a hypothetical library, to prove experimentally selection of smart small-molecule ligands by the proposed approach.

Diffusion as a tool of measuring temperature inside a capillary.

Application of capillary electrophoresis (CE) to temperature-sensitive biomolecular interactions requires knowledge of the temperature inside the capillary. The simplest approach to finding temperature in CE employs a molecular probe with a temperature-dependent parameter. Up until now only spectral parameters of molecular probes were utilized for temperature measurements in CE. The arbitrary nature of spectral parameters leads to several inherent limitations that compromise the accuracy and precision of temperature determination. This paper introduces the concept of finding temperature in CE through the measurement of a nonspectral parameter of the molecular probeits diffusion coefficient. Diffusion is a fundamental property of molecules that depends only on the molecular structure of the probe, the nature of the environment, and the temperature. It is ideally suited for temperature measurements in CE if an approach for measuring the diffusion coefficient in a capillary with high precision is available. This work first develops an approach for measuring the diffusion coefficient in a capillary with a relative standard deviation of as low as 2.1%. It is then demonstrated that such precise measurements of the diffusion coefficient could facilitate accurate temperature determination in CE with a precision of 1 degrees C. This new method was used to study the effect on temperature of different amounts of joule heat generated and different efficiencies of heat dissipation. The nonspectroscopic nature of the method makes it potentially applicable to nonspectroscopic detection schemes, for example, electrochemical and mass spectrometric detection.