Neighboring Base Pairs Research Articles

LncRNATargets: A platform for lncRNA target prediction based on nucleic acid thermodynamics.

Many studies have supported that long noncoding RNAs (lncRNAs) perform various functions in various critical biological processes. Advanced experimental and computational technologies allow access to more information on lncRNAs. Determining the functions and action mechanisms of these RNAs on a large scale is urgently needed. We provided lncRNATargets, which is a web-based platform for lncRNA target prediction based on nucleic acid thermodynamics. The nearest-neighbor (NN) model was used to calculate binging-free energy. The main principle of NN model for nucleic acid assumes that identity and orientation of neighbor base pairs determine stability of a given base pair. lncRNATargets features the following options: setting of a specific temperature that allow use not only for human but also for other animals or plants; processing all lncRNAs in high throughput without RNA size limitation that is superior to any other existing tool; and web-based, user-friendly interface, and colored result displays that allow easy access for nonskilled computer operators and provide better understanding of results. This technique could provide accurate calculation on the binding-free energy of lncRNA-target dimers to predict if these structures are well targeted together. lncRNATargets provides high accuracy calculations, and this user-friendly program is available for free at http://www.herbbol.org:8001/lrt/ .

Entropy Calculations of Hoogsteen and Watson-Crick Conformations

The conformations that DNA takes are often vital in many biological processes. The physical effects Hoogsteen conformation base pairs have on the full strand of DNA are still being determined. Through molecular dynamics simulations in CHARMM, the effect Hoogsteen conformation has on the entropy of the strand can be examined. The absolute entropy of both Hoogsteen and Watson-Crick conformations can be calculated with the use of a quasi-harmonic approximation using the trajectories of each simulation. Using this method, the entropy contribution of varying amounts of neighboring base pairs can be estimated. This work can be compared with experimental entropy values for entire double-strands. Our calculations show how a localized conformational change can affect the entropy in neighboring site and over longer-range base pairs.

Free Energy Calculation for Base Pair Dissociation in a DNA Duplex

DNA is the main genetic material for living organisms including many viruses. DNA duplex, coded with A=T and G=C base pairs, is well suited for biological information storage. The interactions between two bases in a base pair contribute to the stability of DNA duplex, and are further related to gene replication and transcription. In this study, we use all-atom Molecular dynamics (MD) simulations combined with Umbrella sampling (US) method to determine the free energy profiles and explore the molecular details for base pair dissociations. Four groups of DNA duplexes with different sequences have been constructed and a total of 4.3 mu s MD simulations have been carried out. In the potential of mean force (PMF) profile for G equivalent to C base pair dissociation (denoted as PMF-PGC), we observed three peaks, which correspond to the three moments G equivalent to C base pair loses its three hydrogen bonds respectively. Differently, A=T base pair loses its two hydrogen bonds within a very short time. As a result, only one hydrogen bond rupture peak was observed in its PMF curve (denoted as PMF-PAT). Compared with PMF-PAT, the overall free energy barrier in PMF-PGC is higher, which is due to the better stability of G equivalent to C than A=T. In the latter sections of both PMFs, free energies are still increasing, which is mainly resulted from the rigidity of DNA duplex backbone. We have also investigated the impact of neighboring base pairs on the stability of middle one. It is found that neighboring G equivalent to C base pairs increase the stability of A=T base pair while neighboring C equivalent to G base pairs reduce the stability of A=T base pair. Additionally, neighboring T=A base pairs have little influence on the stability of A=T base pair.

Base-Displaced Intercalated Structure of the N-(2'-Deoxyguanosin-8-yl)-3-aminobenzanthrone DNA Adduct.

3-Nitrobenzanthrone (3-NBA), an environmental mutagen found in diesel exhaust and a suspected carcinogen, undergoes metabolic reduction followed by reaction with DNA to form aminobenzanthrone (ABA) adducts, with the major alkylation product being N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (C8-dG-ABA). Site-specific synthesis of the C8-dG-ABA adduct in the oligodeoxynucleotide 5'-d(GTGCXTGTTTGT)-3':5'-d(ACAAACACGCAC)-3'; X = C8-dG-ABA adduct, including codons 272-275 of the p53 gene, has allowed for investigation into the structural and thermodynamic properties of this adduct. The conformation of the C8-dG-ABA adduct was determined using NMR spectroscopy and was refined using molecular dynamics (MD) calculations restrained by experimentally determined interproton distance restraints obtained from NOE experiments. The refined structure revealed that the C8-dG-ABA adduct formed a base-displaced intercalated conformation. The adducted guanine was shifted into the syn conformation about the glycosidic bond. The 5'- and 3'-neighboring base pairs remained intact. While this facilitated π-stacking interactions between the ABA moiety and neighboring bases, the thermal melting temperature (Tm) of the adduct-containing duplex showed a decrease of 11 °C as compared to the corresponding unmodified oligodeoxynucleotide duplex. Overall, in this sequence, the base-displaced intercalated conformation of the C8-dG-ABA lesion bears similarity to structures of other arylamine C8-dG adducts. However, in this sequence, the base-displaced intercalated conformation for the C8-dG-ABA adduct differs from the conformation of the N(2)-dG-ABA adduct reported by de los Santos and co-workers, in which it is oriented in the minor groove toward the 5' end of the duplex, with the modified guanine remaining in the anti conformation about the glyosidic torsion angle, and the complementary base remaining within the duplex. The results are discussed in relationship to differences between the C8-dG-ABA and N(2)-dG-ABA adducts with respect to susceptibility to nucleotide excision repair (NER).

Modulational instability and energy localization of twisted DNA with solvent interaction

We investigate the generation of soliton-like pulses along a DNA chain which takes into account both torsional and solvent interaction effects. Interactions between neighboring base pairs are described by a twist angle. Twisting is essential in the model to capture the importance of nonlinear effects for the thermodynamical properties. The nonlinear dynamics of the DNA is then modeled in the Hamiltonian approach by the generalized Dauxois–Peyrard–Bishop model (combination of several models). We introduce the generalized discrete nonlinear Schrödinger equation describing the dynamics of modulated wave through the twisted DNA with solvent interaction. The modulational instability is studied and we present an analytical expression for the MI gain to show the effects of twist angle on MI gain spectra as well as on stability diagram. With the increase of the twist angle the MI gain decreases then increases. Some interesting MI phenomena appear with an additional new MI region as the twist angle increases. The instability and stability diagrams are also affected. Numerical simulations are carried out to show the validity of the analytical approach. The result is that the initial wave breaks into a train of ultrashort pulses with repetition rates, which are trapped in some sites. The impact of the twist angle is investigated and we obtain that the twist angle affects the dynamics of stable patterns generated through the molecule. Thereafter, we study energy localization in the framework of twisted DNA with solvent interaction. While the twist angle leads to a stronger localization of energy, the solvent interaction delocalizes energy along the molecule.

Stacking geometry for non-canonical G:U wobble base pair containing dinucleotide sequences in RNA: dispersion-corrected DFT-D study.

Emergence of thousands of crystal structures of noncoding RNA molecules indicates its structural and functional diversity. RNA function is based upon a large variety of structural elements which are specifically assembled in the folded molecules. Along with the canonical Watson-Crick base pairs, different orientations of the bases to form hydrogen-bonded non-canonical base pairs have also been observed in the available RNA structures. Frequencies of occurrences of different non-canonical base pairs in RNA indicate their important role to maintain overall structure and functions of RNA. There are several reports on geometry and energetic stabilities of these non-canonical base pairs. However, their stacking geometry and stacking stability with the neighboring base pairs are not well studied. Among the different non-canonical base pairs, the G:U wobble base pair (G:U W:WC) is most frequently observed in the RNA double helices. Using quantum chemical method and available experimental data set we have studied the stacking geometry of G:U W:WC base pair containing dinucleotide sequences in roll-slide parameters hyperspace for different values of twist. This study indicates that the G:U W:WC base pair can stack well with the canonical base pairs giving rise to large interaction energy. The overall preferred stacking geometry in terms of roll, twist and slide for the eleven possible dinucleotide sequences is seen to be quite dependent on their sequences.

Twenty years: a very short sequence in the RNA world.

Lucky for me, my scientific career has encompassed the entire “official” history of the RNA journal and the RNA Society: ribozymes to miRNAs to lncRNAs; Northern blots to PCR to deep sequencing; nuclease structure probing to crystallography to cryo-electron microscopy. Of course, I wasn't there at the very beginning—the RNA world rumbled for quite a while before the Society erupted. At the time, some of the RNA world's rumblings were hard to understand. Why were mutations that affected the function of the ribosome in the RNA, not the protein? What!?—You think you can predict the stability of an RNA stem just by adding the free energies of nearest neighbor base pairs? Why was messenger RNA missing sequences that existed in the DNA encoding it? What ARE all of those small nuclear RNAs? You're telling me it does that without protein! Whoa, RNA-RNA base pairing between those snRNAs and mRNA is required for splicing out those missing pieces!! The various RNA world rumblings ultimately resulted in the RNA Processing meeting, held at Cold Spring Harbor Laboratory (CSHL), and this meeting was the precursor to today's RNA Society meeting. The very first talk I gave in my scientific career was at the RNA Processing meeting in an old CSHL auditorium that is no longer used. I was in a cast, having recently hurt my knee skiing. The weather was cold so I wore long pants over my full-length walking cast, and for years, many RNA scientists thought I had a permanent disability. (At the banquet, the late David Apirion told me sweetly that he didn't know what was wrong with me, but if I had been able to dance, he would have asked me.) The official CSHL pointer at the time was an ∼20-foot-long bamboo pole that tapered at the end. I remember holding on tightly to the podium in hopes that me and my cast would not topple over and watching the end of the pole wave wildly as it transmitted the impulse from my shaking hands. In those days women scientists were fewer, and we were all housed in the “Hooper” dormitory, where Barbara McClintock had an apartment on the ground floor. One morning I came out of the building and there she was, just standing in the yard. I still kick myself that as an inexperienced young graduate student I didn't have the nerve to talk to this legend, since I never was presented with this opportunity again. Hooper was an old building, and typically we did not lock the doors to our dorm rooms; in fact, I don't remember even having a key. However, one year, for reasons that are unclear, something changed. I don't remember whether we were given keys, but I do know that we were not informed that the doors would lock behind us. This of course led to a number of scientists returning to their rooms after their showers, in their towels, only to find that they were locked out of their rooms. For the next part of the story I do not know the truth from the urban legend. But what I heard (amidst lots of giggles) was that when one of our most loved and esteemed Society members was caught in the hall in her “skivvies,” the problem was quickly taken care of by an esteemed CSHL resident who was NOT a member of the RNA Society. What I do know for certain is that when we all returned to our rooms that night, the lock on each and every room was carefully taped to the open position. Yup, there was a lot of history before the RNA Society finally erupted. In fact, the dawn of the RNA Society and its journal was not a precise moment, but began with subtle rumblings that a few, and then many, could feel. (I should note that there are true and indisputable facts in this article, although this is clearly a biased history. While I decided not to use names of the living, suffice it to say, you will know who you are and are welcome to reveal yourself to your colleagues.)

Differential stabilities and sequence-dependent base pair opening dynamics of Watson-Crick base pairs with 5-hydroxymethylcytosine, 5-formylcytosine, or 5-carboxylcytosine.

5-Hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) form during active demethylation of 5-methylcytosine (5mC) and are implicated in epigenetic regulation of the genome. They are differentially processed by thymine DNA glycosylase (TDG), an enzyme involved in active demethylation of 5mC. Three modified Dickerson–Drew dodecamer (DDD) sequences, amenable to crystallographic and spectroscopic analyses and containing the 5′-CG-3′ sequence associated with genomic cytosine methylation, containing 5hmC, 5fC, or 5caC placed site-specifically into the 5′-T8X9G10-3′ sequence of the DDD, were compared. The presence of 5caC at the X9 base increased the stability of the DDD, whereas 5hmC or 5fC did not. Both 5hmC and 5fC increased imino proton exchange rates and calculated rate constants for base pair opening at the neighboring base pair A5:T8, whereas 5caC did not. At the oxidized base pair G4:X9, 5fC exhibited an increase in the imino proton exchange rate and the calculated kop. In all cases, minimal effects to imino proton exchange rates occurred at the neighboring base pair C3:G10. No evidence was observed for imino tautomerization, accompanied by wobble base pairing, for 5hmC, 5fC, or 5caC when positioned at base pair G4:X9; each favored Watson–Crick base pairing. However, both 5fC and 5caC exhibited intranucleobase hydrogen bonding between their formyl or carboxyl oxygens, respectively, and the adjacent cytosine N4 exocyclic amines. The lesion-specific differences observed in the DDD may be implicated in recognition of 5hmC, 5fC, or 5caC in DNA by TDG. However, they do not correlate with differential excision of 5hmC, 5fC, or 5caC by TDG, which may be mediated by differences in transition states of the enzyme-bound complexes.

NMR proton chemical shift prediction of C·C mismatches in B-DNA

Accurate prediction of DNA chemical shifts facilitates resonance assignment and allows recognition of different conformational features. Based on the nearest neighbor model and base pair replacement approach, we have determined a set of triplet chemical shift values and correction factors for predicting the proton chemical shifts of B-DNA containing an internal C·C mismatch. Our results provide a reliable chemical shift prediction with an accuracy of 0.07ppm for non-labile protons and 0.09ppm for labile protons. In addition, we have also shown that the correction factors for C·C mismatches can be used interchangeably with those for T·T mismatches. As a result, we have generalized a set of correction factors for predicting the flanking residue chemical shifts of pyrimidine·pyrimidine mismatches.

Probing the relative orientation of molecules bound to DNA by second-harmonic generation.

We develop a model to probe the relative orientation of two second-order polarizable daunomycin molecules that are intercalated into a DNA duplex using optical second-harmonic (SH) generation. The SH field generated by the daunomycin molecules interfere with each other. Because the interference depends on the relative orientation of the daunomycin molecules, we can control the interference by changing the number of base pairs separating them. The relative orientation changes as the number of base pairs separating them, multiplied by 36°, which is the twist angle between neighboring base pairs. In this paper, we derive a set of relationships between the relative angle of the molecules and the nonlinear susceptibility elements, and we calculate the SH field generated by the DNA/molecular-pair complex attached to an isotropic dielectric sphere. Calculations reveal that the SH intensity varies periodically with the relative orientation of the two chromophores in the plane perpendicular to the helical axis. The predicted periodicity is in close agreement with experimental results. Structural changes induced by foreign molecules binding to DNA will change the relative orientation of the two chromophores and thereby change the SH interference pattern. We discuss the potential of this SH interference method in providing a new way to probe structural changes induced by the formation of biomolecule complexes. An important feature of the method is that it is label-free, that is, the binding molecule, in this case, daunomycin, is not tagged.

Cy3 and Cy5 dyes terminally attached to 5'C end of DNA: structure, dynamics, and energetics.

Cy3 and Cy5 cyanine dyes terminally attached to the 5'C end (C1) of the DNA oligonucleotide were studied by metadynamics (MTD), molecular dynamics (MD), and density-functional methods with dispersion corrections (DFT-D). MTD simulations explored the free energy surface (FES) of the dye-DNA interactions, which included stacking and major groove binding motifs and unstacked structures. Dynamics of the stacked structures was studied by the MD simulations. All possible combinations of stacking interactions between the two indole rings of the dyes and the neighbor guanine and cytosine rings were observed. The most probable interaction included the stacking between the dye's distal indole ring and the guanine base. In ∼10% of the structures the delocalized π-electrons of the dyes' polymethine linkers played a key role in the dye-DNA dispersion interactions. The stacked conformers of the Cy3 dye were confirmed as true minima by DFT-D full optimizations. The stacked dye decreased flexibility up to two neighbor base pairs.

Base Pair Fraying in Molecular Dynamics Simulations of DNA and RNA.

Terminal base pairs of DNA and RNA molecules in solution are known to undergo frequent transient opening events (fraying). Accurate modeling of this process is important because of its involvement in nucleic acid end recognition and enzymatic catalysis. In this article, we describe fraying in molecular dynamics simulations with the ff99bsc0, ff99bsc0χOL3, and ff99bsc0χOL4 force fields, both for DNA and RNA molecules. Comparison with the experiment showed that while some features of fraying are consistent with the available data, others indicate potential problems with the force field description. In particular, multiple noncanonical structures are formed at the ends of the DNA and RNA duplexes. Among them are tWC/sugar edge pair, C-H edge/Watson-Crick pair, and stacked geometries, in which the terminal bases are stacked above each other. These structures usually appear within the first tens to hundreds of nanoseconds and substantially limit the usefulness of the remaining part of the simulation due to geometry distortions that are transferred to several neighboring base pairs ("end effects"). We show that stability of the noncanonical structures in ff99bsc0 may be partly linked to inaccurate glycosidic (χ) torsion potentials that overstabilize the syn region and allow for rapid anti to syn transitions. The RNA refined glycosidic torsion potential χOL3 provides an improved description and substantially more stable MD simulations of RNA molecules. In the case of DNA, the χOL4 correction gives only partial improvement. None of the tested force fields provide a satisfactory description of the terminal regions, indicating that further improvement is needed to achieve realistic modeling of fraying in DNA and RNA molecules.

Optimal guideRNAs for re-directing deaminase activity of hADAR1 and hADAR2 in trans.

Adenosine deaminases that act on RNA (ADAR) are a class of enzymes that catalyze the conversion of adenosine to inosine in RNA. Since inosine is read as guanosine ADAR activity formally introduces A-to-G point mutations. Re-addressing ADAR activity toward new targets in an RNA-dependent manner is a highly rational, programmable approach for the manipulation of RNA and protein function. However, the strategy encounters limitations with respect to sequence and codon contexts. Selectivity is difficult to achieve in adenosine-rich sequences and some codons, like 5′-GAG, seem virtually inert. To overcome such restrictions, we systematically studied the possibilities of activating difficult codons by optimizing the guideRNA that is applied in trans. We find that all 5′-XAG codons with X = U, A, C, G are editable in vitro to a substantial amount of at least 50% once the guideRNA/mRNA duplex is optimized. Notably, some codons, including CAG and GAG, accept or even require the presence of 5′-mismatched neighboring base pairs. This was unexpected from the reported analysis of global editing preferences on large double-stranded RNA substrates. Furthermore, we report the usage of guanosine mismatching as a means to suppress unwanted off-site editing in proximity to targeted adenosine bases. Together, our findings are very important to achieve selective and efficient editing in difficult codon and sequence contexts.

Proximity-induced superconductivity effect in a double-stranded DNA

We study the proximity-induced superconductivity effect in a double-stranded DNA by solving the Bogoliubov-de Gennes equations and taking into account the effect of thermal fluctuations of the twist angle between neighboring base pairs. We show that the electron conductance is spin-dependent and the conductance of spin up (down) increases (decreases) due to the spin-orbit coupling. It is found that, for T<100K, the band gap energy is temperature-independent and it decreases due to the SOC. In addition, by solving the Bogoliubov-de Gennes equations and local gap parameter equation self-consistently, we find the critical temperature at which transition to superconductivity can take place.

Base-displaced intercalation of the 2-amino-3-methylimidazo[4,5-f]quinolone N2-dG adduct in the NarI DNA recognition sequence.

2-Amino-3-methylimidazo[4,5-f]quinolone (IQ), a heterocyclic amine found in cooked meats, undergoes bioactivation to a nitrenium ion, which alkylates guanines at both the C8-dG and N2-dG positions. The conformation of a site-specific N2-dG-IQ adduct in an oligodeoxynucleotide duplex containing the iterated CG repeat restriction site of the NarI endonuclease has been determined. The IQ moiety intercalates, with the IQ H4a and CH3 protons facing the minor groove, and the IQ H7a, H8a and H9a protons facing the major groove. The adducted dG maintains the anti-conformation about the glycosyl bond. The complementary dC is extruded into the major groove. The duplex maintains its thermal stability, which is attributed to stacking between the IQ moiety and the 5′- and 3′-neighboring base pairs. This conformation is compared to that of the C8-dG-IQ adduct in the same sequence, which also formed a ‘base-displaced intercalated’ conformation. However, the C8-dG-IQ adopted the syn conformation placing the Watson−Crick edge of the modified dG into the major groove. In addition, the C8-dG-IQ adduct was oriented with the IQ CH3 group and H4a and H5a facing the major groove. These differences may lead to differential processing during DNA repair and replication.

Site-Specific Stabilization of DNA by a Tethered Major Groove Amine, 7-Aminomethyl-7-deaza-2′-deoxyguanosine

A cationic 7-aminomethyl-7-deaza-2′-deoxyguanosine (7amG) was incorporated site-specifically into the self-complementary duplex d(G1A2G3A4X5C6G7C8T9C10T11C12)2 (X = 7amG). This construct placed two positively charged amines adjacent to the major groove edges of two symmetry-related guanines, providing a model for probing how cation binding in the major groove modulates the structure and stability of DNA. Molecular dynamics calculations restrained by nuclear magnetic resonance (NMR) data revealed that the tethered cationic amines were in plane with the modified base pairs. The tethered amines did not form salt bridges to the phosphodiester backbone. There was also no indication of the amines being capable of hydrogen bonding to flanking DNA bases. NMR spectroscopy as a function of temperature revealed that the X5 imino resonance remained sharp at 55 °C. Additionally, two 5′-neighboring base pairs, A4:T9 and G3:C10, were stabilized with respect to the exchange of their imino protons with solvent. The equilibrium constant for base pair opening at the A4:T9 base pair determined by magnetization transfer from water in the absence and presence of added ammonia base catalyst decreased for the modified duplex compared to that of the A4:T9 base pair in the unmodified duplex, which confirmed that the overall fraction of the A4:T9 base pair in the open state of the modified duplex decreased. This was also observed for the G3:C10 base pair, where αKop for the G3:C10 base pair in the modified duplex was 3.0 × 106 versus 4.1 × 106 for the same base pair in the unmodified duplex. In contrast, equilibrium constants for base pair opening at the X5:C8 and C6:G7 base pairs did not change at 15 °C. These results argue against the notion that electrostatic interactions with DNA are entirely entropic and suggest that major groove cations can stabilize DNA via enthalpic contributions to the free energy of duplex formation.

PT-ACRAMTU, a platinum-acridine anticancer agent, lengthens and aggregates, but does not stiffen or soften DNA.

We used atomic force microscopy (AFM) to study the dose-dependent change in conformational and mechanical properties of DNA treated with PT-ACRAMTU ([PtCl(en)(ACRAMTU-S)](NO3)2, (en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea. PT-ACRAMTU is the parent drug of a family of non-classical platinum-based agents that show potent activity in non-small cell lung cancer in vitro and in vivo. Its acridine moiety intercalates between DNA bases, while the platinum group forms mono-adducts with DNA bases. AFM images show that PT-ACRAMTU causes some DNA looping and aggregation at drug-to-base pair ratio (r b) of 0.1 and higher. Very significant lengthening of the DNA was observed with increasing doses of PT-ACRAMTU, and reached saturation at an r b of 0.15. At r b of 0.1, lengthening was 0.6 nm per drug molecule, which is more than one fully stretched base pair stack can accommodate, indicating that ACRAMTU also disturbs the stacking of neighboring base pair stacks. Analysis of the AFM images based on the worm-like chain (WLC) model showed that PT-ACRAMTU did not change the flexibility of (non-aggregated) DNA, despite the extreme lengthening. The persistence length of untreated DNA and DNA treated with PT-ACRAMTU was in the range of 49-65 nm. Potential consequences of the perturbations caused by this agent for the recognition and processing of the DNA adducts it forms are discussed.

Mechanism for Template-Independent Terminal Adenylation Activity of Qβ Replicase

The genomic RNA of Qβ virus is replicated by Qβ replicase, a template-dependent RNA polymerase complex. Qβ replicase has an intrinsic template-independent RNA 3'-adenylation activity, which is required for efficient viral RNA amplification in thehost cells. However, the mechanism of the template-independent 3'-adenylation of RNAs by Qβ replicase has remained elusive. We determined the structure of a complex that includes Qβ replicase, a template RNA, a growing RNA complementary to the template RNA, and ATP. The structure represents the terminal stage of RNA polymerization and reveals that the shape and size of the nucleotide-binding pocket becomes available for ATP accommodation after the 3'-penultimate template-dependent C-addition. The stacking interaction between the ATP and the neighboring Watson-Crick base pair, between the 5'-G in the template and the 3'-C in the growing RNA, contributes to the nucleotide specificity. Thus, the template for the template-independent 3'-adenylation by Qβ replicase is the RNA and protein ribonucleoprotein complex.

Sequence-dependent Structural Variation in DNA Undergoing Intrahelical Inspection by the DNA glycosylase MutM

MutM, a bacterial DNA-glycosylase, plays a critical role in maintaining genome integrity by catalyzing glycosidic bond cleavage of 8-oxoguanine (oxoG) lesions to initiate base excision DNA repair. The task faced by MutM of locating rare oxoG residues embedded in an overwhelming excess of undamaged bases is especially challenging given the close structural similarity between oxoG and its normal progenitor, guanine (G). MutM actively interrogates the DNA to detect the presence of an intrahelical, fully base-paired oxoG, whereupon the enzyme promotes extrusion of the target nucleobase from the DNA duplex and insertion into the extrahelical active site. Recent structural studies have begun to provide the first glimpse into the protein-DNA interactions that enable MutM to distinguish an intrahelical oxoG from G; however, these initial studies left open the important question of how MutM can recognize oxoG residues embedded in 16 different neighboring sequence contexts (considering only the 5'- and 3'-neighboring base pairs). In this study we set out to understand the manner and extent to which intrahelical lesion recognition varies as a function of the 5'-neighbor. Here we report a comprehensive, systematic structural analysis of the effect of the 5'-neighboring base pair on recognition of an intrahelical oxoG lesion. These structures reveal that MutM imposes the same extrusion-prone ("extrudogenic") backbone conformation on the oxoG lesion irrespective of its 5'-neighbor while leaving the rest of the DNA relatively free to adjust to the particular demands of individual sequences.

Effects of Non-Nearest Neighbors on the Thermodynamic Stability of RNA GNRA Hairpin Tetraloops

Currently, several models for predicting the secondary structure of RNA exist, one of which is free energy minimization using the Nearest Neighbor Model. This model predicts the lowest-free energy secondary structure from a primary sequence by summing the free energy contributions of the Watson-Crick nearest neighbor base pair combinations and any noncanonical secondary structure motif. The Nearest Neighbor Model also assumes that the free energy of the secondary structure motif is dependent solely on the identities of the nucleotides within the motif and the motif's nearest neighbors. To test the current assumption of the Nearest Neighbor Model that the non-nearest neighbors do not affect the stability of the motif, we optically melted different stem-loop oligonucleotides to experimentally determine their thermodynamic parameters. In each of these oligonucleotides, the hairpin loop sequence and the adjacent base pairs were held constant, while the first or second non-nearest neighbors were varied. The experimental results show that the thermodynamic contributions of the hairpin loop were dependent upon the identity of the first non-nearest neighbor, while the second non-nearest neighbor had a less obvious effect. These results were then used to create an updated model for predicting the thermodynamic contributions of a hairpin loop to the overall stability of the stem-loop structure.