A study of 7-deaza-2'-deoxyguanosine 2'-deoxycytidine base pairing in DNA.
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ABSTRACT:
SUBMITTER: Ganguly M
PROVIDER: S-EPMC2094092 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:As part of an ongoing effort to explore the effect of major groove electrostatics on the thermodynamic stability and structure of DNA, a 7-deaza-2'-deoxyadenosine:dT (7-deaza-dA:dT) base pair in the Dickerson-Drew dodecamer (DDD) was studied. The removal of the electronegative N7 atom on dA and the replacement with an electropositive C-H in the major groove was expected to have a significant effect on major groove electrostatics. The structure of the 7-deaza-dA:dT base pair was determined at 1.1 Å resolution in the presence of Mg(2+). The 7-deaza-dA, which is isosteric for dA, had minimal effect on the base pairing geometry and the conformation of the DDD in the crystalline state. There was no major groove cation association with the 7-deaza-dA heterocycle. In solution, circular dichroism showed a positive Cotton effect centered at 280 nm and a negative Cotton effect centered at 250 nm that were characteristic of a right-handed helix in the B-conformation. However, temperature-dependent NMR studies showed increased exchange between the thymine N3 imino proton of the 7-deaza-dA:dT base pair and water, suggesting reduced stacking interactions and an increased rate of base pair opening. This correlated with the observed thermodynamic destabilization of the 7-deaza-dA modified duplex relative to the DDD. A combination of UV melting and differential scanning calorimetry experiments were conducted to evaluate the relative contributions of enthalpy and entropy in the thermodynamic destabilization of the DDD. The most significant contribution arose from an unfavorable enthalpy term, which probably results from less favorable stacking interactions in the modified duplex, which was accompanied by a significant reduction in the release of water and cations from the 7-deaza-dA modified DNA.
| S-EPMC3225014 | biostudies-literature
Project description:Bacterial production has been often estimated from DNA synthesis rates by using tritium-labeled thymidine. Some bacteria species cannot incorporate extracellular thymidine into their DNA, suggesting their biomass production might be overlooked when using the conventional method. In the present study, to evaluate appropriateness of deoxyribonucleosides for evaluating bacterial production of natural bacterial communities from the viewpoint of DNA synthesis, incorporation rates of four deoxyribonucleosides (thymidine, deoxyadenosine, deoxyguanosine and deoxycytidine) labeled by nitrogen stable isotope (15N) into bacterial DNA were examined in both ocean (Sagami Bay) and freshwater (Lake Kasumigaura) ecosystems in July 2015 and January 2016. In most stations in Sagami Bay and Lake Kasumigaura, we found that incorporation rates of deoxyguanosine were the highest among those of the four deoxyribonucleosides, and the incorporation rate of deoxyguanosine was approximately 2.5 times higher than that of thymidine. Whereas, incorporation rates of deoxyadenosine and deoxycytidine were 0.9 and 0.2 times higher than that of thymidine. These results clearly suggest that the numbers of bacterial species which can incorporate exogenous deoxyguanosine into their DNA are relatively greater as compared to the other deoxyribonucleosides, and measurement of bacterial production using deoxyguanosine more likely reflects larger numbers of bacterial species productions.
| S-EPMC7046229 | biostudies-literature
Project description: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 αK(op) 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.
| S-EPMC3812902 | biostudies-literature
Project description:Several reports have demonstrated that Campylobacter bacteriophage DNA is refractory to manipulation, suggesting that these phages encode modified DNA. The characterized Campylobacter jejuni phages fall into two phylogenetic groups within the Myoviridae: the genera Firehammervirus and Fletchervirus Analysis of genomic nucleosides from several of these phages by high-pressure liquid chromatography-mass spectrometry confirmed that 100% of the 2'-deoxyguanosine (dG) residues are replaced by modified bases. Fletcherviruses replace dG with 2'-deoxyinosine, while the firehammerviruses replace dG with 2'-deoxy-7-amido-7-deazaguanosine (dADG), noncanonical nucleotides previously described, but a 100% base substitution has never been observed to have been made in a virus. We analyzed the genome sequences of all available phages representing both groups to elucidate the biosynthetic pathway of these noncanonical bases. Putative ADG biosynthetic genes are encoded by the Firehammervirus phages and functionally complement mutants in the Escherichia coli queuosine pathway, of which ADG is an intermediate. To investigate the mechanism of DNA modification, we isolated nucleotide pools and identified dITP after phage infection, suggesting that this modification is made before nucleotides are incorporated into the phage genome. However, we were unable to observe any form of dADG phosphate, implying a novel mechanism of ADG incorporation into an existing DNA strand. Our results imply that Fletchervirus and Firehammervirus phages have evolved distinct mechanisms to express dG-free DNA.IMPORTANCE Bacteriophages are in a constant evolutionary struggle to overcome their microbial hosts' defenses and must adapt in unconventional ways to remain viable as infectious agents. One mode of adaptation is modifying the viral genome to contain noncanonical nucleotides. Genome modification in phages is becoming more commonly reported as analytical techniques improve, but guanosine modifications have been underreported. To date, two genomic guanosine modifications have been observed in phage genomes, and both are low in genomic abundance. The significance of our research is in the identification of two novel DNA modification systems in Campylobacter-infecting phages, which replace all guanosine bases in the genome in a genus-specific manner.
| S-EPMC6854489 | biostudies-literature
Project description:8-Oxo-7,8-dihydroguanine (OG) is the most common base damage found in cells, where it resides in many structural contexts, including the nucleotide pool, single-stranded DNA at transcription forks and replication bubbles, and duplex DNA base-paired with either adenine (A) or cytosine (C). OG is prone to further oxidation to the highly mutagenic hydantoin products spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) in a sharply pH-dependent fashion within nucleosides. In the present work, studies were conducted to determine how the structural context affects OG oxidation to the hydantoins. These studies revealed a trend in which the Sp yield was greatest in unencumbered contexts, such as nucleosides, while the Gh yield increased in oligodeoxynucleotide (ODN) contexts or at reduced pH. Oxidation of oligomers containing hydrogen-bond modulators (2,6-diaminopurine, N(4)-ethylcytidine) or alteration of the reaction conditions (pH, temperature, and salt) identify base stacking, electrostatics, and base pairing as the drivers of the key intermediate 5-hydroxy-8-oxo-7,8-dihydroguanine (5-HO-OG) partitioning along the two hydantoin pathways, allowing us to propose a mechanism for the observed base-pairing effects. Moreover, these structural effects cause an increase in the effective pK(a) of 5-HO-OG, following an increasing trend from 5.7 in nucleosides to 7.7 in a duplex bearing an OG·C base pair, which supports the context-dependent product yields. The high yield of Gh in ODNs underscores the importance of further study on this lesion. The structural context of OG also determined its relative reactivity toward oxidation, for which the OG·A base pair is ~2.5-fold more reactive than an OG·C base pair, and with the weak one-electron oxidant ferricyanide, the OG nucleoside reactivity is >6000-fold greater than that of OG·C in a duplex, leading to the conclusion that OG in the nucleoside pool should act as a protective agent for OG in the genome.
| S-EPMC3440533 | biostudies-literature
Project description:The replacement of the 7-N atom on guanine (G) with a C-H to give 7-deazaguanine (c(7)G) alters the electronic properties of the heterocyclic base and eliminates a potential major groove cation binding site, which affects the organization of salts and water in the major groove. This has a destabilizing effect on DNA. We report herein the characterization of DNA oligomers containing 7-(aminomethyl)-7-deazaguanine (1) residues using a variety of spectroscopic and thermodynamic approaches. 1 is an intramolecular model for the major groove binding of cations and basic amino acid residues to G. In contrast to c(7)G, the tethering of a cation in the major groove using 1 affords DNA that is as, or more, stable than the corresponding unmodified DNA. The stabilization is associated with the folding enthalpy and hydration.
| S-EPMC2767112 | biostudies-literature
Project description:Hoogsteen (HG) base pairing is characterized by a 180° rotation of the purine base with respect to the Watson-Crick-Franklin (WCF) motif. Recently, it has been found that both conformations coexist in a dynamical equilibrium and that several biological functions require HG pairs. This relevance has motivated experimental and computational investigations of the base-pairing transition. However, a systematic simulation of sequence variations has remained out of reach. Here, we employ advanced path-based methods to perform unprecedented free-energy calculations. Our methodology enables us to study the different mechanisms of purine rotation, either remaining inside or after flipping outside of the double helix. We study seven different sequences, which are neighbor variations of a well-studied A⋅T pair in A6-DNA. We observe the known effect of A⋅T steps favoring HG stability, and find evidence of triple-hydrogen-bonded neighbors hindering the inside transition. More importantly, we identify a dominant factor: the direction of the A rotation, with the 6-ring pointing either towards the longer or shorter segment of the chain, respectively relating to a lower or higher barrier. This highlights the role of DNA's relative flexibility as a modulator of the WCF/HG dynamic equilibrium. Additionally, we provide a robust methodology for future HG proclivity studies.
| S-EPMC9177043 | biostudies-literature
Project description:CpG islands are GC rich sequences that are found in the promoters of many genes in higher eukaryotes. They contain a high frequency of CG dinucleotides, which are substrates for DNA methylases. Methylation leads to transcriptional silencing of promoters. Owing to their high GC content CpG islands exhibit strong base-base interactions, which lead to superstructures and consequently to regions with higher melting temperatures. Therefore, Taq polymerases (especially sequenases) fall off their templates, causing premature termination of the polymerase chain reaction (PCR) or sequencing reactions. The results from such reactions are thus insufficient for further analysis. Therefore, we have evaluated the use of 7-deaza-2'-deoxyguanosine for PCR amplification of the human p16(INK4A) promoter and sequencing of HUMARA exon 1 PCR products. Our results show that the addition of 7-deaza-2'-deoxyguanosine significantly improves results, particularly when small amounts of poor quality DNA are available as starting material.
| S-EPMC1187147 | biostudies-literature
Project description:The Hoogsteen (HG) base pairing conformation, commonly observed in damaged and mutated DNA helices, facilitates DNA repair and DNA recognition. The free energy difference between HG and Watson-Crick (WC) base pairs has been computed in previous studies. However, the mechanism of the conformational transition is not well understood. A detailed understanding of the process of WC to HG base pair transition can provide a deeper understanding of DNA repair and recognition. In an earlier study, we explored the free energy landscape for this process using extensive computer simulation with the CHARMM36 force field. In this work, we study the impact of force field models in describing the WC to HG base pairing transition using meta-eABF enhanced sampling, quasi-harmonic entropy calculation, and nonbonded energy analysis. The secondary structures of both base pairing forms and the topology of the free energy landscapes were consistent over different force field models, although the relative free energy, entropy, and the interaction energies tend to vary. The relative stability of the WC and HG conformations is dictated by a delicate balance between the enthalpic stabilization and the reduced entropy of the structurally rigid HG structure. These findings highlight the impact that subtleties in force field models can have on accurately modeling DNA base pair dynamics and should stimulate further computational investigations into other dynamically important motions in DNA.
| S-EPMC9795553 | biostudies-literature
Project description:Two factors are mainly responsible for the stability of the DNA double helix: base pairing between complementary strands and stacking between adjacent bases. By studying DNA molecules with solitary nicks and gaps we measure temperature and salt dependence of the stacking free energy of the DNA double helix. For the first time, DNA stacking parameters are obtained directly (without extrapolation) for temperatures from below room temperature to close to melting temperature. We also obtain DNA stacking parameters for different salt concentrations ranging from 15 to 100 mM Na+. From stacking parameters of individual contacts, we calculate base-stacking contribution to the stability of A*T- and G*C-containing DNA polymers. We find that temperature and salt dependences of the stacking term fully determine the temperature and the salt dependence of DNA stability parameters. For all temperatures and salt concentrations employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A*T pairing is always destabilizing and G*C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence.
| S-EPMC1360284 | biostudies-literature