ABSTRACT
The formation of a stable G-quadruplex (GQ) can inhibit the increased telomerase activity that is common in most cancers. The global structure and the thermal stability of the GQs are usually evaluated by spectroscopic methods and thermal denaturation properties. However, most biochemical processes involving GQs might require local conformational changes at the guanine tetrad (G4) level. These local conformational changes of individual G4 layers during protein and drug interactions have not yet been explored in detail. In this study, we monitored the local conformations of individual G4 layers in GQs using 6-methylisoxanthopterine (6MI) chromophores, which are circular dichroism (CD)-active fluorescent base analogues of guanine, as local conformational probes. A synthetic, tetramolecular, parallel GQ with site-specifically positioned 6MI monomers or dimers was used as the experimental construct. Analytical ultracentrifugation studies and gel electrophoretic studies showed that properly positioned 6MI monomers and dimers could form stable GQs with CD-active fluorescent G4 layers. The local conformation of individual fluorescent G4 layers in the GQ structure was then tracked by monitoring the absorbance, fluorescence intensity, thermal melting, fluorescence quenching, and CD changes of the incorporated probes. Overall, these studies showed that site-specifically incorporated fluorescent base analogues could be used as probes to monitor the local conformational changes of individual G4 layers of a GQ structure. This method can be applied to explore the details of small molecule-GQ interaction at the level of the individual G4 layers, which may prove to be useful in designing drugs to treat GQ-related genetic disorders, cancer, and aging.
Subject(s)
G-Quadruplexes , Telomerase , Circular Dichroism , Guanine/chemistryABSTRACT
We previously demonstrated that flavonoid metabolites inhibit cancer cell proliferation through both CDK-dependent and -independent mechanisms. The existing evidence suggests that gut microbiota is capable of flavonoid biotransformation to generate bioactive metabolites including 2,4,6-trihydroxybenzoic acid (2,4,6-THBA), 3,4-dihydroxybenzoic acid (3,4-DHBA), 3,4,5-trihyroxybenzoic acid (3,4,5-THBA) and 3,4-dihydroxyphenylacetic acid (DOPAC). In this study, we screened 94 human gut bacterial species for their ability to biotransform flavonoid quercetin into different metabolites. We demonstrated that five of these species were able to degrade quercetin including Bacillus glycinifermentans, Flavonifractor plautii, Bacteroides eggerthii, Olsenella scatoligenes and Eubacterium eligens. Additional studies showed that B. glycinifermentans could generate 2,4,6-THBA and 3,4-DHBA from quercetin while F. plautii generates DOPAC. In addition to the differences in the metabolites produced, we also observed that the kinetics of quercetin degradation was different between B. glycinifermentans and F. plautii, suggesting that the pathways of degradation are likely different between these strains. Similar to the antiproliferative effects of 2,4,6-THBA and 3,4-DHBA demonstrated previously, DOPAC also inhibited colony formation ex vivo in the HCT-116 colon cancer cell line. Consistent with this, the bacterial culture supernatant of F. plautii also inhibited colony formation in this cell line. Thus, as F. plautii and B. glycinifermentans generate metabolites possessing antiproliferative activity, we suggest that these strains have the potential to be developed into probiotics to improve human gut health.
Subject(s)
3,4-Dihydroxyphenylacetic Acid/pharmacology , Antineoplastic Agents/pharmacology , Bacteria/classification , Bromobenzoates/pharmacology , Gallic Acid/pharmacology , Hydroxybenzoates/pharmacology , Quercetin/chemistry , 3,4-Dihydroxyphenylacetic Acid/chemistry , Actinobacteria/genetics , Actinobacteria/isolation & purification , Actinobacteria/metabolism , Antineoplastic Agents/chemistry , Bacillus/genetics , Bacillus/isolation & purification , Bacillus/metabolism , Bacteria/genetics , Bacteria/isolation & purification , Bacteria/metabolism , Bacterial Proteins , Bacteroides/genetics , Bacteroides/isolation & purification , Bacteroides/metabolism , Bromobenzoates/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Clostridiales/genetics , Clostridiales/isolation & purification , Clostridiales/metabolism , Eubacterium/genetics , Eubacterium/isolation & purification , Eubacterium/metabolism , Gallic Acid/chemistry , Gastrointestinal Microbiome , Gene Expression Profiling , Gene Expression Regulation, Bacterial , HCT116 Cells , Humans , Hydroxybenzoates/chemistry , Phylogeny , Sequence Analysis, RNAABSTRACT
In this study, we use single-stranded DNA (oligo-dT) lattices that have been position-specifically labeled with monomer or dimer 2-aminopurine (2-AP) probes to map the local interactions of the DNA bases with the nucleic acid binding cleft of gp32, the single-stranded binding (ssb) protein of bacteriophage T4. Three complementary spectroscopic approaches are used to characterize these local interactions of the probes with nearby nucleotide bases and amino acid residues at varying levels of effective protein binding cooperativity, as manipulated by changing lattice length. These include: (i) examining local quenching and enhancing effects on the fluorescence spectra of monomer 2-AP probes at each position within the cleft; (ii) using acrylamide as a dynamic-quenching additive to measure solvent access to monomer 2-AP probes at each ssDNA position; and (iii) employing circular dichroism spectra to characterize changes in exciton coupling within 2-AP dimer probes at specific ssDNA positions within the protein cleft. The results are interpreted in part by what we know about the topology of the binding cleft from crystallographic studies of the DNA binding domain of gp32 and provide additional insights into how gp32 can manipulate the ssDNA chain at various steps of DNA replication and other processes of genome expression.
Subject(s)
Bacteriophage T4/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Nucleic Acid Conformation , Viral Proteins/metabolism , 2-Aminopurine/chemistry , Binding Sites , Circular Dichroism , Crystallography, X-Ray , DNA, Single-Stranded/chemistry , Fluorescent Dyes , Protein Binding , Spectrometry, FluorescenceABSTRACT
Labeling substrates or products are paramount in determining enzymatic kinetic parameters. Several options are available; many laboratories use either radioactive or fluorescent labeling because of their high sensitivity. However, those methods have their own drawbacks such as half-life decay, expensive and hazardous. Here, we propose a novel, simple, economical and fast alternative to substrate labeling for studying the kinetics of nucleic acids: post-migration gel staining with SYBR Gold. Cleavage rates similar to the ones reported in the literature for the I-R3 DNA-cleaving DNA enzyme in the presence of zinc chloride are an indication of the quality of the new method. Moreover, the activity of the hammerhead ribozyme was also monitored by our method to illustrate its versatility. This labeling-free method has several advantages such as its ease of use as well as cost effective and versatility with both non-structured and structured RNAs or DNAs.
Subject(s)
Fluorescent Dyes , Nucleic Acids/analysis , Organic Chemicals , 2-Aminopurine/chemistry , Binding Sites , Chlorides/metabolism , DNA/analysis , DNA/genetics , DNA, Catalytic/metabolism , Kinetics , Nucleic Acid Conformation , Nucleic Acids/genetics , RNA, Catalytic/metabolism , Spectrometry, Fluorescence , Staining and Labeling/methods , Substrate Specificity , Zinc Compounds/metabolismABSTRACT
DNA replication is a core biological process that occurs in prokaryotic cells at high speeds (â¼1 nucleotide residue added per millisecond) and with high fidelity (fewer than one misincorporation event per 107 nucleotide additions). The ssDNA binding protein [gene product 32 (gp32)] of the T4 bacteriophage is a central integrating component of the replication complex that must continuously bind to and unbind from transiently exposed template strands during DNA synthesis. We here report microsecond single-molecule FRET (smFRET) measurements on Cy3/Cy5-labeled primer-template (p/t) DNA constructs in the presence of gp32. These measurements probe the distance between Cy3/Cy5 fluorophores that label the ends of a short (15-nt) segment of ssDNA attached to a model p/t DNA construct and permit us to track the stochastic interconversion between various protein bound and unbound states. The length of the 15-nt ssDNA lattice is sufficient to accommodate up to two cooperatively bound gp32 proteins in either of two positions. We apply a unique multipoint time correlation function analysis to the microsecond-resolved smFRET data obtained to determine and compare the kinetics of various possible reaction pathways for the assembly of cooperatively bound gp32 protein onto ssDNA sequences located at the replication fork. The results of our analysis reveal the presence and translocation mechanisms of short-lived intermediate bound states that are likely to play a critical role in the assembly mechanisms of ssDNA binding proteins at replication forks and other ss duplex junctions.
Subject(s)
Bacteriophage T4/chemistry , DNA, Single-Stranded/chemistry , DNA-Binding Proteins/chemistry , Fluorescence Resonance Energy Transfer , Viral Proteins/chemistry , Bacteriophage T4/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Viral Proteins/metabolismABSTRACT
Se pretende determinar la prevalencia de los diferentes trastornos psicológicos en una muestra clínica de niños y adolescentes (0 a 14 años) valorados en Pediatría de Atención Primaria y el efecto del sexo y la edad. Se realiza un estudio descriptivo con el total de las valoraciones realizadas (712) entre los años 2013 a 2015. El sexo y la edad son variables que determinan la presencia o ausencia de los trastornos. Los trastornos que presentan un incremento con la edad dependiendo del sexo son los trastornos afectivos, los de ansiedad, los de conducta y los de déficit de atención con hiperactividad (TDAH). Estos resultados permitirán una adecuación de los programas de coordinación, colaboración y atención a la población infantil y juvenil, así como la adaptación de los programas formativos
Effect of sex and age. This study aims to determine the prevalence of different psychological disorders in a clinical sample of children and adolescents (0-14 years of age) who were assessed at Primary Care Pediatric examining the effects of sex and age. A descriptive study was carried out with all the assessments (712) made between the years of 2013 and 2015. Sex and age are variables that determine the presence or absence of disorders. Disorders that show an increase with age depending on sex include affective disorders, anxiety, behavior disorders and attention deficit hyperactivity disorder (ADHD). These results allow for the adaptation of coordination and collaboration programs and aimed at children and the young population child and youth population to better suit the needs of these groups, and for the adaptation of training programs
Efecte del sexe i ledat. Es pretén determinar la prevalença dels diferents trastorns psicològics en una mostra clínica de nens i adolescents (0 a 14 anys) valorats a pediatria datenció primària i lefecte del sexe i de ledat. Es realitza un estudi descriptiu amb el total de les valoracions realitzades (712) entre els anys 2013 a 2015. El sexe i ledat són variables que determinen la presència o absència dels trastorns. Els trastorns que presenten un increment amb ledat depenent del sexe són els trastorns afectius, els dansietat, els de conducta i els de dèficit datenció amb hiperactivitat. Aquests resultats permetran una adequació dels programes de coordinació, col·laboració i atenció a la població infantil i juvenil, així com ladaptació dels programes formatius
Subject(s)
Humans , Child , Adolescent , Neurodevelopmental Disorders/epidemiology , Mental Disorders/epidemiology , 50293 , Sex Factors , Affective Symptoms/epidemiology , Anxiety Disorders/epidemiology , Attention Deficit Disorder with Hyperactivity/epidemiology , Child Behavior Disorders/epidemiology , Retrospective StudiesABSTRACT
Gene 32 protein (gp32) is the single-stranded (ss) DNA binding protein of the bacteriophage T4. It binds transiently and cooperatively to ssDNA sequences exposed during the DNA replication process and regulates the interactions of the other sub-assemblies of the replication complex during the replication cycle. We here use single-molecule FRET techniques to build on previous thermodynamic studies of gp32 binding to initiate studies of the dynamics of the isolated and cooperative binding of gp32 molecules within the replication complex. DNA primer/template (p/t) constructs are used as models to determine the effects of ssDNA lattice length, gp32 concentration, salt concentration, binding cooperativity and binding polarity at p/t junctions. Hidden Markov models (HMMs) and transition density plots (TDPs) are used to characterize the dynamics of the multi-step assembly pathway of gp32 at p/t junctions of differing polarity, and show that isolated gp32 molecules bind to their ssDNA targets weakly and dissociate quickly, while cooperatively bound dimeric or trimeric clusters of gp32 bind much more tightly, can 'slide' on ssDNA sequences, and exhibit binding dynamics that depend on p/t junction polarities. The potential relationships of these binding dynamics to interactions with other components of the T4 DNA replication complex are discussed.
Subject(s)
Bacteriophage T4 , DNA, Single-Stranded/chemistry , DNA-Binding Proteins/chemistry , Viral Proteins/chemistry , DNA Replication , DNA, Viral/chemistry , Fluorescence Resonance Energy Transfer , Kinetics , Protein Binding , Sodium Chloride/chemistry , Virus ReplicationABSTRACT
Combining biophysical measurements on T4 bacteriophage replication complexes with detailed structural information can illuminate the molecular mechanisms of these 'macromolecular machines'. Here we use the low energy circular dichroism (CD) and fluorescent properties of site-specifically introduced base analogues to map and quantify the equilibrium binding interactions of short (8 nts) ssDNA oligomers with gp32 monomers at single nucleotide resolution. We show that single gp32 molecules interact most directly and specifically near the 3'-end of these ssDNA oligomers, thus defining the polarity of gp32 binding with respect to the ssDNA lattice, and that only 2-3 nts are directly involved in this tight binding interaction. The loss of exciton coupling in the CD spectra of dimer 2-AP (2-aminopurine) probes at various positions in the ssDNA constructs, together with increases in fluorescence intensity, suggest that gp32 binding directly extends the sugar-phosphate backbone of this ssDNA oligomer, particularly at the 3'-end and facilitates base unstacking along the entire 8-mer lattice. These results provide a model (and 'DNA map') for the isolated gp32 binding to ssDNA targets, which serves as the nucleation step for the cooperative binding that occurs at transiently exposed ssDNA sequences within the functioning T4 DNA replication complex.
Subject(s)
DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Viral Proteins/metabolism , 2-Aminopurine , Binding Sites , Circular Dichroism , DNA, Single-Stranded/chemistry , DNA-Binding Proteins/chemistry , Nucleotides/chemistry , Protein Binding , Protein Multimerization , Viral Proteins/chemistryABSTRACT
We here use our site-specific base analog mapping approach to study the interactions and binding equilibria of cooperatively-bound clusters of the single-stranded DNA binding protein (gp32) of the T4 DNA replication complex with longer ssDNA (and dsDNA) lattices. We show that in cooperatively bound clusters the binding free energy appears to be equi-partitioned between the gp32 monomers of the cluster, so that all bind to the ssDNA lattice with comparable affinity, but also that the outer domains of the gp32 monomers at the ends of the cluster can fluctuate on and off the lattice and that the clusters of gp32 monomers can slide along the ssDNA. We also show that at very low binding densities gp32 monomers bind to the ssDNA lattice at random, but that cooperatively bound gp32 clusters bind preferentially at the 5'-end of the ssDNA lattice. We use these results and the gp32 monomer-binding results of the companion paper to propose a detailed model for how gp32 might bind to and interact with ssDNA lattices in its various binding modes, and also consider how these clusters might interact with other components of the T4 DNA replication complex.
Subject(s)
DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Viral Proteins/metabolism , 2-Aminopurine , Binding Sites , Circular Dichroism , DNA Replication , DNA, Single-Stranded/chemistry , Fluorescent Dyes , Models, Biological , Nucleotides/chemistry , Protein Binding , Thermodynamics , Xanthopterin/analogs & derivativesABSTRACT
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
Subject(s)
Ultracentrifugation/methods , Ultracentrifugation/standards , Calibration , Reproducibility of ResultsABSTRACT
DNA "breathing" is a thermally driven process in which base-paired DNA sequences transiently adopt local conformations that depart from their most stable structures. Polymerases and other proteins of genome expression require access to single-stranded DNA coding templates located in the double-stranded DNA "interior," and it is likely that fluctuations of the sugar-phosphate backbones of dsDNA that result in mechanistically useful local base pair opening reactions can be exploited by such DNA regulatory proteins. Such motions are difficult to observe in bulk measurements, both because they are infrequent and because they often occur on microsecond time scales that are not easy to access experimentally. We report single-molecule fluorescence experiments with polarized light, in which tens-of-microseconds rotational motions of internally labeled iCy3/iCy5 donor-acceptor Förster resonance energy transfer fluorophore pairs that have been rigidly inserted into the backbones of replication fork constructs are simultaneously detected using single-molecule Förster resonance energy transfer and single-molecule fluorescence-detected linear dichroism signals. Our results reveal significant local motions in the â¼100-µs range, a reasonable time scale for DNA breathing fluctuations of potential relevance for DNA-protein interactions. Moreover, we show that both the magnitudes and the relaxation times of these backbone breathing fluctuations are significantly perturbed by interactions of the fork construct with a nonprocessive, weakly binding bacteriophage T4-coded helicase hexamer initiation complex, suggesting that these motions may play a fundamental role in the initial binding, assembly, and function of the processive helicase-primase (primosome) component of the bacteriophage T4-coded DNA replication complex.
Subject(s)
DNA Replication , DNA, Viral/metabolism , Fluorescence Resonance Energy Transfer/methods , Viral Proteins/metabolism , Algorithms , Bacteriophage T4/genetics , Bacteriophage T4/metabolism , Carbocyanines/chemistry , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA, Viral/chemistry , DNA, Viral/genetics , Fluorescent Dyes/chemistry , Kinetics , Models, Genetic , Models, Molecular , Nucleic Acid Conformation , Protein Multimerization , Viral Proteins/chemistry , Viral Proteins/geneticsABSTRACT
Single-molecule fluorescence resonance energy transfer (smFRET) methods were used to study the assembly pathway and DNA unwinding activity of the bacteriophage T4 helicase-primase (primosome) complex. The helicase substrates used were surface-immobilized model DNA replication forks "internally" labeled in the duplex region with opposed donor/acceptor (iCy3/iCy5) chromophore pairs in the lagging and leading strands. The time dependence of the smFRET signals was monitored during the unwinding process, and helicase rates and processivities were measured as a function of GTP concentration. This smFRET approach was also used to investigate the subunit stoichiometry of the primosome and the assembly pathway required to form functional and fully active primosome-DNA complexes. We confirmed that gp41 helicase monomer subunits form stable hexameric helicases in the presence of GTP and that the resulting (gp41)(6) complexes bind only weakly at DNA fork junctions. The addition of a single subunit of gp61 primase stabilized the resulting primosome complex at the fork and resulted in fully active and processive primosome helicases with gp41:gp61 subunit ratios of 6:1, while higher and lower subunit ratios substantially reduced the primosome unwinding activity. The use of alternative assembly pathways resulted in a loss of helicase activity and the formation of metastable DNA-protein aggregates, which were easily detected in our smFRET experiments as intense light-scattering foci. These single-molecule experiments provide a detailed real-time visualization of the assembly pathway and duplex DNA unwinding activity of the T4 primosome and are consistent with more indirect equilibrium and steady state results obtained in bulk solution studies.
Subject(s)
Bacteriophage T4/enzymology , DNA Helicases/metabolism , DNA Primase/metabolism , Adenosine Triphosphatases/metabolism , DNA/chemistry , DNA/metabolism , Fluorescence Resonance Energy Transfer , Models, Molecular , ProbabilityABSTRACT
Physical biochemical techniques are used to establish the structure, subunit stoichiometry, and assembly pathway of the primosome complex of the bacteriophage T4 DNA replication system. Analytical ultracentrifugation and fluorescence anisotropy methods show that the functional T4 primosome consists of six gp41 helicase subunits that assemble into a hexagon, driven by the binding of six NTPs (or six nonhydrolyzable GTPγS analogues) that are located at and stabilize the intersubunit interfaces, together with a single tightly bound gp61 primase subunit. Assembling the components of the primosome onto a model DNA replication fork is a multistep process, but equilibrium cannot be reached along all mixing pathways. Producing a functional complex requires that the helicase hexamer be assembled in the presence of the DNA replication fork construct prior to the addition of the primase to avoid the formation of metastable DNA-protein aggregates. The gp41 helicase hexamer binds weakly to fork DNA in the absence of primase, but forms a much more stable primosome complex that expresses full and functional helicase (and primase) activities when bound to a gp61 primase subunit at a helicase:primase subunit ratio of 61. The presence of additional primase subunits does not change the molecular mass or helicase activity of the primosome, but significantly inhibits its primase activity. We develop both an assembly pathway and a minimal mechanistic model for the structure and function of the T4 primosome that are likely to be relevant to the assembly and function of the replication primosome subassemblies of higher organisms as well.
Subject(s)
Bacteriophage T4/chemistry , DNA Helicases/chemistry , DNA Primase/chemistry , Anisotropy , Bacterial Proteins/chemistry , DNA/chemistry , DNA Replication , Dose-Response Relationship, Drug , Guanosine 5'-O-(3-Thiotriphosphate)/chemistry , Guanosine Triphosphate/chemistry , Hydrolysis , Macromolecular Substances/chemistry , Microscopy, Fluorescence/methods , Models, Chemical , Protein Denaturation , Thermodynamics , Ultracentrifugation/methods , Viral Envelope Proteins/metabolismABSTRACT
We previously used changes in the near-UV circular dichroism and fluorescence spectra of DNA base analogue probes placed site specifically to show that the first three base pairs at the fork junction in model replication fork constructs are significantly opened by "breathing" fluctuations under physiological conditions. Here, we use these probes to provide mechanistic snapshots of the initial interactions of the DNA fork with a tight-binding replication helicase in solution. The primosome helicase of bacteriophage T4 was assembled from six (gp41) helicase subunits, one (gp61) primase subunit, and nonhydrolyzable GTPγS. When bound to a DNA replication fork construct this complex advances one base pair into the duplex portion of the fork and forms a stably bound helicase "initiation complex." Replacement of GTPγS with GTP permits the completion of the helicase-driven unwinding process. Our spectroscopic probes show that the primosome in this stable helicase initiation complex binds the DNA of the fork primarily via backbone contacts and holds the first complementary base pair of the fork in an open conformation, whereas the second, third, and fourth base pairs of the duplex show essentially the breathing behavior that previously characterized the first three base pairs of the free fork. These spectral changes, together with dynamic fluorescence quenching results, are consistent with a primosome-binding model in which the lagging DNA strand passes through the central hole of the hexagonal helicase, the leading strand binds to the "outside" surfaces of subunits of the helicase hexamer, and the single primase subunit interacts with both strands.
Subject(s)
Bacteriophage T4/physiology , Base Pairing/genetics , DNA Helicases/metabolism , DNA Replication/physiology , Nucleic Acid Conformation , Bacteriophage T4/enzymology , Circular Dichroism , DNA Replication/genetics , Models, Molecular , Spectrometry, FluorescenceABSTRACT
Junctions between ssDNA and dsDNA sequences are important in many cellular processes, including DNA replication, transcription, recombination, and repair. Significant transient conformational fluctuations ("DNA breathing") can occur at these ssDNA-dsDNA junctions. The involvement of such breathing in the mechanisms of macromolecular complexes that operate at these loci is not well understood, in part because these fluctuations have been difficult to measure in a position-specific manner. To address this issue we constructed forked or primer-template DNA constructs with 1 or 2 adjacent 2-aminopurine (2-AP) nucleotide residues (adenine analogues) placed at specific positions on both sides of the ssDNA-dsDNA junction. Unlike canonical DNA bases, 2-AP absorbs, fluoresces, and displays CD spectra at wavelengths >300 nm, where other nucleic acid and protein components are transparent. We used CD and fluorescence spectra and acrylamide quenching of these probes to monitor the extent and nature of DNA breathing of A-T base pairs at specific positions around the ssDNA-dsDNA junction. As expected, spectroscopically measurable unwinding penetrates approximately 2 bp into the duplex region of these junctions under physiological conditions for the constructs examined. Surprisingly, we found that 2-AP bases at ssDNA sites directly adjacent to ssDNA-dsDNA junctions are significantly more unstacked than those at more distant ssDNA positions. These local and transient DNA conformations on both sides of ssDNA-dsDNA junctions may serve as specific interaction targets for enzymes that manipulate DNA in the processes of gene expression.
Subject(s)
DNA Replication , DNA/chemistry , DNA Primers , DNA, Single-Stranded , Nucleic Acid Conformation , Spectrum Analysis , Templates, Genetic , VibrationABSTRACT
Although mechanisms of single-nucleotide residue deletion have been investigated, processes involved in the loss of longer nucleotide sequences during DNA replication are poorly understood. Previous reports have shown that in vitro replication of a 3'-TGC TGC template sequence can result in the deletion of one 3'-TGC. We have used low-energy circular dichroism (CD) and fluorescence spectroscopy to investigate the conformations and stabilities of DNA models of the replication intermediates that may be implicated in this frameshift. Pyrrolocytosine or 2-aminopurine residues, site-specifically substituted for cytosine or adenine in the vicinity of extruded base sequences, were used as spectroscopic probes to examine local DNA conformations. An equilibrium mixture of four hybridization conformations was observed when template bases looped-out as a bulge, i.e. a structure flanked on both sides by duplex DNA. In contrast, a single-loop structure with an unusual unstacked DNA conformation at its downstream edge was observed when the extruded bases were positioned at the primer-template junction, showing that misalignments can be modified by neighboring DNA secondary structure. These results must be taken into account in considering the genetic and biochemical mechanisms of frameshift mutagenesis in polymerase-driven DNA replication.
Subject(s)
DNA Replication , DNA/chemistry , Frameshift Mutation , Circular Dichroism , DNA Primers , Models, Genetic , Nucleic Acid Conformation , Sequence Deletion , Spectrometry, Fluorescence , Templates, GeneticABSTRACT
The dynamics of the B-A transition of DNA double helices with different GC contents and various chain lengths has been characterized by an electric field pulse technique. The field-induced B-A reaction is separated from orientation effects using the magic angle technique. Amplitudes reflecting the B-A reaction are observed selectively in the limited range of ethanol contents, where CD spectra demonstrate the B-A transition. The maximum amplitude appears at 1-2% higher ethanol content than the center of the B-A transition observed by CD because electric field pulses induce a relatively large perturbation from the A- toward the B-form. The relaxation curves measured after pulse termination reflect a spectrum of up to three relaxation processes. For DNA's with approximately 50% GC, the main part of the amplitude ( approximately 75%) is associated with time constants of approximately 2 micros, and another major component appears with time constants of 50-100 micros. These relaxation effects have been observed for DNA samples with 859, 2629, 7160, and 48501 bp. The time constant associated with the main amplitude increases with decreasing GC content from approximately 2 micros at 50% GC to approximately 3 mus at 41% GC and approximately 10 micros at 0% GC at the center of the B-A transition. Model calculations on the kinetics of cooperative linear Ising lattices predict the appearance of a distinct maximum of the mean relaxation time at the center of the transition. The absence of such maximum in our experimental data indicates a low cooperativity of the B-A transition with a nucleation parameter of approximately 0.1. The rate of the B-A transition is lower by approximately 3 orders of magnitude than that predicted by molecular dynamics simulations.
Subject(s)
DNA, A-Form/chemistry , DNA/chemistry , Nucleic Acid Conformation , Animals , Bacteriophage lambda/genetics , Base Composition , DNA/genetics , DNA, A-Form/genetics , DNA, Viral/chemistry , DNA, Viral/genetics , Electrons , Male , Plasmids/chemistry , Plasmids/genetics , Poly dA-dT/chemistry , Poly dA-dT/genetics , Salmon/genetics , Spectrum Analysis , TestisABSTRACT
Although the transition from the B-DNA double helix to the A-form is essential for biological function, as shown by the existence of the A-form in many protein-DNA complexes, the dynamics of this transition has not been resolved yet. According to molecular dynamics simulations the transition is expected in the time range of a few nanoseconds. The B-A transition induced by mixing of DNA samples with ethanol in stopped flow experiments is complete within the deadtime, showing that the reaction is faster than approximately 0.2 ms. The reaction was resolved by an electric field jump technique with induction of the transition by a dipole stretching force driving the A- to the B-form. Poly[d(A-T)] was established as a favourable model system, because of a particularly high cooperativity of the transition and because of a spectral signature allowing separation of potential side reactions. The time constants observed in the case of poly[d(A-T)] with approximately 1600 bp are in the range around 10 micros. An additional process with time constants of approximately 100 micros is probably due to nucleation. The same time constants (within experimental accuracy +/-10%) were observed for a poly[d(A-T)] sample with approximately 70 bp. Under low salt conditions commonly used for studies of the B-A transition, the time constants are almost independent of the ionic strength. The experimental data show that a significant activation barrier exists in the B-A transition and that the helical states are clearly separated from each other, in contrast to predictions by molecular dynamics simulations.