DNA Base Flipping Research Articles

Can specific THz fields induce collective base-flipping in DNA? A stochastic averaging and resonant enhancement investigation based on a new mesoscopic model.

We study the metastability, internal frequencies, activation mechanism, energy transfer, and the collective base-flipping in a mesoscopic DNA via resonance with specific electric fields. Our new mesoscopic DNA model takes into account not only the issues of helicity and the coupling of an electric field with the base dipole moments, but also includes environmental effects, such as fluid viscosity and thermal noise. Also, all the parameter values are chosen to best represent the typical values for the opening and closing dynamics of a DNA. Our study shows that while the mesoscopic DNA is metastable and robust to environmental effects, it is vulnerable to certain frequencies that could be targeted by specific THz fields for triggering its collective base-flipping dynamics and causing large amplitude separation of base pairs. Based on applying the Freidlin-Wentzell method of stochastic averaging and the newly developed theory of resonant enhancement to our mesoscopic DNA model, our semi-analytic estimates show that the required fields should be THz fields with frequencies around 0.28 THz and with amplitudes in the order of 450 kV/cm. These estimates compare well with the experimental data of Titova et al., which have demonstrated that they could affect the function of DNA in human skin tissues by THz pulses with frequencies around 0.5 THz and with a peak electric field at 220 kV/cm. Moreover, our estimates also conform to a number of other experimental results, which appeared in the last couple years.

Uracil-DNA glycosylase efficiency is modulated by substrate rigidity.

DNA sequence effects have been identified for many glycosylases and alkyl-transferases that repair base mismatches, uracil bases, alkylated bases, etc. Here, we focused on understanding how DNA sequence impacts the repair of uracil, a highly mutagenic and common lesion in DNA. Uracil DNA-glycosylase (UNG) is a base excision repair enzyme that removes uracil from DNA by a base flipping mechanism. UNG excision efficiency depends on DNA sequence. We show that UNG efficiency is dictated by the intrinsic local deformability of the substrate sequence around the uracil. UNG specificity constants (kcat/KM) and DNA flexibilities were measured for an engineered set of DNA substrates. Time-resolved fluorescence spectroscopy, NMR imino proton exchange measurements, and molecular dynamics simulations of the bare DNA indicated significant differences in substrate flexibilities. We observed a strong correlation between UNG efficiency and substrate flexibility, with higher kcat/KM values measured for more flexible strands. DNA bending and base flipping were observed in simulations, with more frequent uracil flipping observed for the more bendable sequences. Experiments show that bases immediately adjacent to the uracil are allosterically coupled and have the greatest impact on substrate flexibility and resultant UNG activity. A better understanding of the fundamental principles that dictate UNG activity has broad implications in diverse fields, from cancer to evolution.

Extension of the CHARMM Classical Drude Polarizable Force Field to N- and O-Linked Glycopeptides and Glycoproteins.

Molecular dynamic simulations are an effective tool to study complex molecular systems and are contingent upon the availability of an accurate and reliable molecular mechanics force field. The Drude polarizable force field, which allows for the explicit treatment of electronic polarization in a computationally efficient fashion, has been shown to reproduce experimental properties that were difficult or impossible to reproduce with the CHARMM additive force field, including peptide folding cooperativity, RNA hairpin structures, and DNA base flipping. Glycoproteins are essential components of glycoconjugate vaccines, antibodies, and many pharmaceutically important molecules, and an accurate polarizable force field that includes compatibility between the protein and carbohydrate aspect of the force field is essential to study these types of systems. In this work, we present an extension of the Drude polarizable force field to glycoproteins, including both N- and O-linked species. Parameter optimization focused on the dihedral terms using a reweighting protocol targeting NMR solution J-coupling data for model glycopeptides. Validation of the model include eight model glycopeptides and four glycoproteins with multiple N- and O-linked glycosylations. The new glycoprotein carbohydrate force field can be used in conjunction with the remainder of Drude polarizable force field through a variety of MD simulation programs including GROMACS, OPENMM, NAMD, and CHARMM and may be accessed through the Drude Prepper module in the CHARMM-GUI.

Recent Advances on DNA Base Flipping: A General Mechanism for Writing, Reading, and Erasing DNA Modifications.

The modification of DNA bases is a classic hallmark of epigenetics. Four forms of modified cytosine-5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine-have been discovered in eukaryotic DNA. In addition to cytosine carbon-5 modifications, cytosine and adenine methylated in the exocyclic amine-N4-methylcytosine and N6-methyladenine-are other modified DNA bases discovered even earlier. Each modified base can be considered a distinct epigenetic signal with broader biological implications beyond simple chemical changes. Since 1994, several crystal structures of proteins and enzymes involved in writing, reading, and erasing modified bases have become available. Here, we present a structural synopsis of writers, readers, and erasers of the modified bases from prokaryotes and eukaryotes. Despite significant differences in structures and functions, they are remarkably similar regarding their engagement in flipping a target base/nucleotide within DNA for specific recognitions and/or reactions. We thus highlight base flipping as a common structural framework broadly applied by distinct classes of proteins and enzymes across phyla for epigenetic regulations of DNA.

DNA sequence-dependent activity and base flipping mechanisms of DNMT1 regulate genome-wide DNA methylation

DNA methylation maintenance by DNMT1 is an essential process in mammals but molecular mechanisms connecting DNA methylation patterns and enzyme activity remain elusive. Here, we systematically analyzed the specificity of DNMT1, revealing a pronounced influence of the DNA sequences flanking the target CpG site on DNMT1 activity. We determined DNMT1 structures in complex with preferred DNA substrates revealing that DNMT1 employs flanking sequence-dependent base flipping mechanisms, with large structural rearrangements of the DNA correlating with low catalytic activity. Moreover, flanking sequences influence the conformational dynamics of the active site and cofactor binding pocket. Importantly, we show that the flanking sequence preferences of DNMT1 highly correlate with genomic methylation in human and mouse cells, and 5-azacytidine triggered DNA demethylation is more pronounced at CpG sites with flanks disfavored by DNMT1. Overall, our findings uncover the intricate interplay between CpG-flanking sequence, DNMT1-mediated base flipping and the dynamic landscape of DNA methylation.

Free Energy Coupling between DNA Bending and Base Flipping.

Free energy simulations are presented to probe the energetic coupling between DNA bending and the flipping of a central thymine in double stranded DNA 13mers. The energetics are shown to depend on the neighboring base pairs, and upstream C or T or downstream C tended to make flipping more costly. Flipping to the major groove side was generally preferred. Bending aids flipping, by pushing the system up in free energy, but for small and intermediate bending angles the two were uncorrelated. At higher bending angles, bending and flipping became correlated, and bending primed the system for base flipping toward the major groove. Flipping of the 6-4 pyrimidine-pyrimidone and pyrimidine dimer photoproducts is shown to be more facile than for undamaged DNA. For the damages, major groove flipping was preferred, and DNA bending was much facilitated in the 6-4 pyrimidine-pyrimidone damaged system. Aspects of the calculations were verified by structural analyses of protein-DNA complexes with flipped bases.

DNA Base Flipping: A General Mechanism for Writing, Reading, and Erasing DNA Modifications.

The modification of DNA bases is a classic hallmark of epigenetics. Four forms of modified cytosine-5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine-have been discovered in eukaryotic DNA. In addition to cytosine carbon-5 modifications, cytosine and adenine methylated in the exocyclic amine-N4-methylcytosine and N6-methyladenine-are other modified DNA bases discovered even earlier. Each modified base can be considered a distinct epigenetic signal with broader biological implications beyond simple chemical changes. Since 1994, crystal structures of proteins and enzymes involved in writing, reading, and erasing modified bases have become available. Here, we present a structural synopsis of writers, readers, and erasers of the modified bases from prokaryotes and eukaryotes. Despite significant differences in structures and functions, they are remarkably similar regarding their engagement in flipping a target base/nucleotide within DNA for specific recognitions and/or reactions. We thus highlight base flipping as a common structural framework broadly applied by distinct classes of proteins and enzymes across phyla for epigenetic regulations of DNA.

Influence of DNA Conformation and Repair Enzyme on Guanine and 8-Oxoguanine Base Flipping

8-oxoguanine (8oxoG) is an abundant product of oxidative damage in DNA and if not repaired can result in mutations upon DNA replication. It is removed by repair glycosylases (e.g. MutM-glycosylase in bacteria) after flipping out the damaged base towards an extra-helical conformation into the repair enzyme active site. The exact mechanism how the repair enzyme identifies a damaged site within a large surplus of undamaged DNA is not fully understood. Binding of a repair enzyme results also in significant DNA deformation such as bending and minor groove opening. Looping out nucleotides from an intra-helical base paired conformation is energetically costly and it is not clear how the presence of a repair enzyme or the deformation of DNA may facilitate the looping out process. In this study we use Molecular Dynamics free energy simulations to evaluate the effects of DNA deformation and enzyme binding on the DNA base flipping process. The simulations indicate distinct free energy profiles for flipping 8oxoG or guanine and resulted in an overall calculated free energy for the flipping process in accordance with experimental imino proton exchange from Nuclear Magnetic Resonance spectroscopy. Distortion of the DNA towards a conformation as observed in complex with the repair enzyme lowered the free energy barrier and penalty for the flipping process. The result indicates that the DNA deformation induced by the repair enzyme binding has a significant influence on the flipping process. The additional effect of protein-DNA contacts on the calculated free energy for the flipping process will also be presented.

Switching on the fluorescence of 2-aminopurine by site-selective microhydration.

2-Aminopurine (2 AP) is a fluorescent isomer of adenine and has a fluorescence lifetime of ~11 ns in water. It is widely used in biochemical settings as a site-specific fluorescent probe of DNA and RNA structure and base-flipping and -folding. These assays assume that 2 AP is intrinsically strongly fluorescent. Here, we show this not to be the case, observing that gas-phase, jet-cooled 2-aminopurine and 9-methyl-2-aminopurine have very short fluorescence lifetimes (156 ps and 210 ps, respectively); they are, to all intents and purposes, non-fluorescent. We find that the lifetime of 2-aminopurine increases dramatically when it is part of a hydrate cluster, 2 AP · (H2O)n, where n = 1-3. Not only does it depend on the presence of water molecules, it also depends on the specific hydrogen-bonding site to which they attach and on the number of H2O molecules at that site. We selectively microhydrate 2-aminopurine at its sugar-edge, cis-amino or trans-amino sites and see that its fluorescence lifetime increases by 4, 50 and 95 times (to 14.5 ns), respectively.

Phage Orf Family Recombinases: Conservation of Activities and Involvement of the Central Channel in DNA Binding

Genetic and biochemical evidence suggests that λ Orf is a recombination mediator, promoting nucleation of either bacterial RecA or phage Redβ recombinases onto single-stranded DNA (ssDNA) bound by SSB protein. We have identified a diverse family of Orf proteins that includes representatives implicated in DNA base flipping and those fused to an HNH endonuclease domain. To confirm a functional relationship with the Orf family, a distantly-related homolog, YbcN, from Escherichia coli cryptic prophage DLP12 was purified and characterized. As with its λ relative, YbcN showed a preference for binding ssDNA over duplex. Neither Orf nor YbcN displayed a significant preference for duplex DNA containing mismatches or 1-3 nucleotide bulges. YbcN also bound E. coli SSB, although unlike Orf, it failed to associate with an SSB mutant lacking the flexible C-terminal tail involved in coordinating heterologous protein-protein interactions. Residues conserved in the Orf family that flank the central cavity in the λ Orf crystal structure were targeted for mutagenesis to help determine the mode of DNA binding. Several of these mutant proteins showed significant defects in DNA binding consistent with the central aperture being important for substrate recognition. The widespread conservation of Orf-like proteins highlights the importance of targeting SSB coated ssDNA during lambdoid phage recombination.

Induced Polarization Influences the Fundamental Forces in DNA Base Flipping.

Base flipping in DNA is an important process involved in genomic repair and epigenetic control of gene expression. The driving forces for these processes are not fully understood, especially in the context of the underlying dynamics of the DNA and solvent effects. We studied double-stranded DNA oligomers that have been previously characterized by imino proton exchange NMR using both additive and polarizable force fields. Our results highlight the importance of induced polarization on the base flipping process, yielding near-quantitative agreement with experimental measurements of the equilibrium between the base-paired and flipped states. Further, these simulations allow us to quantify for the first time the energetic implications of polarization on the flipping pathway. Free energy barriers to base flipping are reduced by changes in dipole moments of both the flipped bases that favor solvation of the bases in the open state and water molecules adjacent to the flipping base.

2-Aminopurine Single-Molecule Fluorescence

Base flipping is a local conformational change that results in the rotation of a nucleotide out of its helical structure. This is frequently observed in numerous nucleic acid structural rearrangements, such as in DNA-protein interactions and in different RNA enzymes. 2-Aminopurine (2AP) is a fluorescent nucleotide analog extensively used to probe local conformational changes in nucleic acids in bulk experiments, but no single molecule approaches have been developed to study these mechanisms using 2AP. One challenge towards this goal is that 2AP fluorescence is very sensitive to the interaction with neighboring nucleotides, and its emission spectrum partially overlaps with that of tryptophan. We have developed new click-chemistry-based surface immobilization approach that enables us to monitor, in real time base flipping in DNA. We have characterized the fluorescence properties of single 2AP labelled DNAs. Our results show that nucleotides are very dynamic in a single- or double-stranded DNA and that bases can flip out of the helix, suggesting that the base flipping occurs frequently and in a much slower time scale that previously believe. This new assay can also be used to study other local conformational changes in nucleic acids at the single-molecule level by using 2AP as the base flipping probe.

DNA Base Flipping: New-Found Insights into the DNA Mismatch Recognition Process in E. Coli Muts

Detection of various base-base mismatches and small insertion/deletion loops in DNA is performed by the MutS mismatch recognition protein. This highly conserved process exists in both prokaryotes and eukaryotes and failure to recognize DNA damage can have a detrimental effect on the fidelity of the genome and, in humans, has been linked to numerous forms of cancer. Several crystal structures have emerged over the years capturing MutS bound to various mismatches. However, the events directly following mismatch recognition remain unclear. To shed light on this matter, we present nine sub-μs, all-atom molecular dynamics simulations of Escherichia coli MutS bound to a G•T mismatch with different nucleotide configurations. From these simulations, we identified significant instability in the adjacent base 5′ to the thymine mismatch. In one case, the 5′ adjacent base completely loses base stacking and flips out spontaneously via the minor groove. To the best of our knowledge, this rare event is the first ever documented case of DNA base flipping from any unrestrained protein-DNA simulation. In addition, these observations are in excellent agreement with a recent experimental study where it was suggested that the 5′ adjacent base could exist in an extrahelical state. To further understand the energetics of base flipping in MutS, we utilized the Hamiltonian replica-exchange molecular dynamics (HREMD) method simulating 44 independent replicas in an explicit water box. The free energy profile generated from HREMD shows two distinct minima, one for the stacked state and one for flipped out state, separated by a small energy barrier with the flipped out state being the more favorable of the two. Together, our results offer an unprecedented level of new insight into the mismatch recognition process and further our knowledge of this complex system.

Folding kinetics of recognition loop peptides from a photolyase and cryptochrome-DASH

Cryptochromes (CRY) and photolyases (PL) use a common flavin adenine dinucleotide cofactor and homologous protein scaffold to accomplish numerous, seemingly dissimilar functions. PL repairs UV-damaged DNA in a mechanism requiring light and DNA base flipping. CRY cannot repair DNA, and instead function in core biological processes including plant photomorphogenesis, circadian rhythm, and magnetoreception. One subclass, CRY-DASH, does catalyze repair of single-stranded DNA; compromised base flipping may deactivate its tight binding to duplex DNA substrates. We recently demonstrated that the a “recognition loop” involved in DNA binding by both PL and CRY-DASH is among the most flexible regions in the two proteins, and exhibits especially heightened dynamics in CRY-DASH. Here, we establish that these distinct dynamics are encoded by the loop sequences: we quantify the flexibility of the isolated loop peptides through the kinetics and activation parameters for their folding. Mirroring the dynamics within the proteins, the CRY-DASH recognition loop peptide folds 2.5-fold faster than its counterpart in PL, predominantly due to a lower enthalpy of activation. We propose that these distinct dynamics are functionally significant in DNA recognition. Binding duplex DNA in the catalytically-active base-flipped conformation imposes significant order on the recognition loop, and a corresponding entropic penalty. This may be surmounted by the more preorganized PL recognition loop, but may impose too large a barrier for the more dynamic loop in CRY-DASH. These results suggest that evolution of protein dynamics, through local sequence tuning in the recognition loop, may be an important mechanism for functional diversification in PL and CRY.

Kinetic Mechanism for the Flipping and Excision of 1,N6-Ethenoadenine by Human Alkyladenine DNA Glycosylase

Human alkyladenine DNA glycosylase initiates the repair of a wide variety of alkylated and deaminated purine lesions in DNA. In this study, we take advantage of the natural fluorescence of the 1,N(6)-ethenoadenosine (epsilonA) lesion and report a kinetic analysis of binding, nucleotide flipping, base excision, and product release. The transient changes in the fluorescence of epsilonA revealed the existence of two distinct complexes that are formed prior to the hydrolysis step. An initial recognition complex forms rapidly and is characterized by partial disruption of the stacking interactions of the lesioned base. Subsequently, a very stable extrahelical complex is formed in which the epsilonA lesion is strongly quenched by interactions in the AAG active site pocket. Our results indicate that DNA binding and base flipping take place on the millisecond to second time scale. N-Glycosidic bond cleavage is much slower, taking place on the minute time scale. A pulse-chase experiment was used to demonstrate that even for the tightly bound epsilonA substrate, the extrahelical complex is not fully committed to excision. Nevertheless, flipping of epsilonA is highly favorable, and we calculate that the equilibrium constant for flipping is approximately 1300. This kinetic mechanism has important biological implications. First, two-step binding provides multiple opportunities to discriminate between damaged and undamaged nucleotides. Second, a rapid equilibrium flipping mechanism maximizes specificity for damaged versus undamaged bases, since undamaged bases generally form stronger base pairs than damaged bases. Finally, the highly favorable equilibrium for flipping of epsilonA ensures that epsilonA removal is independent of sequence context and highly efficient despite the relatively slow rate of N-glycosidic bond hydrolysis.

Distinct recognition loop dynamics in cryptochrome-DASH and photolyase revealed by limited proteolysis

Cryptochromes (CRY) are light-responsive flavoproteins that play central roles in nature and human health, including circadian rhythm regulation. They are closely related to photolyases (PL), but, unlike PL, they cannot repair cyclobutane pyrimidine dimers (CPD) or [6–4] photoproducts in duplex DNA. Yet, if the barrier for flipping the CPD from the duplex is reduced, CRY-DASH, the subclass most structurally homologous to CPD PL, binds and repairs CPD like PL. Here, using limited proteolysis, we have identified the most flexible loops in CPD PL. One corresponds to a “recognition loop” that changes conformation substantially during substrate binding, and engages key interactions with the flipped CPD and the complementary DNA strand. Proteolysis kinetics reveal that the homologous loop in CRY-DASH is at least 10-fold more reactive. We propose that heightened dynamics of the recognition loop in CRY-DASH contribute to its compromised DNA base flipping, and its evolution of divergent function from PL.

DNA base flipping by both members of the PspGI restriction-modification system

The PspGI restriction–modification system recognizes the sequence CCWGG. R.PspGI cuts DNA before the first C in the cognate sequence and M.PspGI is thought to methylate N4 of one of the cytosines in the sequence. M.PspGI enhances fluorescence of 2-aminopurine in DNA if it replaces the second C in the sequence, while R.PspGI enhances fluorescence when the fluorophore replaces adenine in the central base pair. This strongly suggests that the methyltransferase flips the second C in the recognition sequence, while the endonuclease flips both bases in the central base pair out of the duplex. M.PspGI is the first N4-cytosine MTase for which biochemical evidence for base flipping has been presented. It is also the first type IIP methyltransferase whose catalytic activity is strongly stimulated by divalent metal ions. However, divalent metal ions are not required for its base-flipping activity. In contrast, these ions are required for both base flipping and catalysis by the endonuclease. The two enzymes have similar temperature profiles for base flipping and optimal flipping occurs at temperatures substantially below the growth temperature of the source organism for PspGI and for the catalytic activity of endonuclease. We discuss the implications of these results for DNA binding by these enzymes and their evolutionary origin.

Differential stabilization of reaction intermediates: specificity checkpoints for M.EcoRI revealed by transient fluorescence and fluorescence lifetime studies

M.EcoRI, a bacterial sequence-specific S-adenosyl-l-methionine-dependent DNA methyltransferase, relies on a complex conformational mechanism to achieve its remarkable specificity, including DNA bending, base flipping and intercalation into the DNA. Using transient fluorescence and fluorescence lifetime studies with cognate and noncognate DNA, we have characterized several reaction intermediates involving the WT enzyme. Similar studies with a bending-impaired, enhanced-specificity M.EcoRI mutant show minimal differences with the cognate DNA, but significant differences with noncognate DNA. These results provide a plausible explanation of the way in which destabilization of reaction intermediates can lead to changes in substrate specificity.

Sequence dependence in base flipping: experimental and computational studies

Base flipping is the movement of a DNA base from an intrahelical, base-stacked position to an extrahelical, solvent-exposed position. As there are favorable interactions for an intrahelical base, both hydrogen bonding and base stacking, base flipping is expected to be energetically prohibitive for an undamaged DNA duplex. For damaged DNA bases, however, the energetic cost of base flipping may be considerably lower. Using a selective, non-covalent assay for base flipping, the sequence dependence of base flipping in DNA sequences containing an abasic site has been studied. The dissociation constants of the zinc-cyclen complex to small molecules and single strands of DNA as well as the equilibrium constants for base flipping have been determined for these sequences. Molecular dynamics simulations of the zinc-cyclen complex bound to both single- and double-stranded DNA have been performed in an attempt to rationalize the differences in the dissociation constants obtained for the two systems. The results are compared to previous studies of base flipping in DNA containing an abasic site.

A rapid reaction analysis of uracil DNA glycosylase indicates an active mechanism of base flipping

Uracil DNA glycosylase (UNG) is the primary enzyme for the removal of uracil from the genome of many organisms. A key question is how the enzyme is able to scan large quantities of DNA in search of aberrant uracil residues. Central to this is the mechanism by which it flips the target nucleotide out of the DNA helix and into the enzyme-active site. Both active and passive mechanisms have been proposed. Here, we report a rapid kinetic analysis using two fluorescent chromophores to temporally resolve DNA binding and base-flipping with DNA substrates of different sequences. This study demonstrates the importance of the protein–DNA interface in the search process and indicates an active mechanism by which UNG glycosylase searches for uracil residues.