Initiation of SV40 DNA replication after microinjection into Xenopus eggs

We have examined the capacity of Xenopus laevis eggs to support replication of microinjected SV40 DNA. As previously reported, microinjected DNA undergoes semi-conservative replication. Unlabeled SV40 DNA was microinjected with [³H]dTTP and, after a 3 h incubation period, the DNA was recovered and adsorbed to BND-cellulose. Elution with an NaCl gradient removes molecules that are entirely double-stranded but not those with single-stranded regions. The latter DNA population is eluted with caffeine. The radioactive DNA that eluted with NaCl was comprised mostly of supercoiled and open circular SV40 DNAs. The radioactive DNA eluted with caffeine was comprised mainly of endogenous DNA but also contained replicative forms of SV40 DNA. Analysis of SV40 DNA replication intermediates by electron microscopy revealed mainly Cairn's forms of varying degrees of maturation. Digestion with BamH1, which cleaves SV40 DNA almost opposite the normal viral replication origin, indicated that SV40 DNA microinjected into frog eggs does not initiate DNA synthesis at its normal initiation site nor at any other obvious preferred site. Rather, it appears that when this template is injected into activated Xenopus eggs, replication may initiate at random.     

Calculation of the minimum energy conformation of biomolecules using a global optimization technique I. Methodology and application to a model molecular fragment (Normal pentane)

The conformational energy of a molecule is minimized with respect to interatomic distances using Bremermann's method of unconstrained global optimization (1970). The optimal set of distances is then used for calculating the preferred conformation of the molecule. A simultaneous optimization of all the dihedral angles is achieved. The classical potential function is used in this study. An illustration of the method is given by applying it to normal pentane, which is a commonly occurring fragment of biomolecules. Results show that, for the standard geometry (bond lengths and bond angles), the all-trans) conformation is the preferred one. However, fluctuations of the geometry within the limits of the vibrational spectra can lead to preferred conformations that are not necessarily all-trans.     

Assembly of ordered DNA-curli fibril complexes during Salmonella biofilm formation correlates with strengths of the type I interferon and autoimmune responses

Deposition of human amyloids is associated with complex human diseases such as Alzheimer’s and Parkinson’s. Amyloid proteins are also produced by bacteria. The bacterial amyloid curli, found in the extracellular matrix of both commensal and pathogenic enteric bacterial biofilms, forms complexes with extracellular DNA, and recognition of these complexes by the host immune system may initiate an autoimmune response. Here, we isolated early intermediate, intermediate, and mature curli fibrils that form throughout the biofilm development and investigated the structural and pathogenic properties of each. Early intermediate aggregates were smaller than intermediate and mature curli fibrils, and circular dichroism, tryptophan, and thioflavin T analyses confirmed the establishment of a beta-sheet secondary structure as the curli conformations matured. Intermediate and mature curli fibrils were more immune stimulatory than early intermediate fibrils in vitro. The intermediate curli was cytotoxic to macrophages independent of Toll-like receptor 2. Mature curli fibrils had the highest DNA content and induced the highest levels of Isg15 expression and TNFα production in macrophages. In mice, mature curli fibrils induced the highest levels of anti-double-stranded DNA autoantibodies. The levels of autoantibodies were higher in autoimmune-prone NZBWxF/1 mice than wild-type C57BL/6 mice. Chronic exposure to all curli forms led to significant histopathological changes and synovial proliferation in the joints of autoimmune-prone mice; mature curli was the most detrimental. In conclusion, curli fibrils, generated during biofilm formation, cause pathogenic autoimmune responses that are stronger when curli complexes contain higher levels of DNA and in mice predisposed to autoimmunity.     

How DNA Polymerase X Preferentially Accommodates Incoming dATP Opposite 8-Oxoguanine on the Template

The modified base 8-oxo-7,8-dihydro-2′-deoxyguanosine (oxoG) is a common DNA adduct produced by the oxidation of DNA by reactive oxygen species. Kinetic data reveal that DNA polymerase X (pol X) from the African swine fever virus incorporates adenine (dATP) opposite to oxoG with higher efficiency than the non-damaged G:C basepair. To help interpret the kinetic data, we perform molecular dynamics simulations of pol X/DNA complexes, in which the template base opposite to the incoming dNTP (dCTP, dATP, dGTP) is oxoG. Our results suggest that pol X accommodates the oxoG_syn:A mispair by sampling closed active conformations that mirror those observed in traditional Watson-Crick complexes. Moreover, for both the oxoG_syn:A and oxoG:C ternary complexes, conformational sampling of the polymerase follows previously described large subdomain movements, local residue motions, and active site reorganization. Interestingly, the oxoG_syn:A system exhibits superior active site geometry in comparison to the oxoG:C system. Simulations for the other mismatch basepair complexes reveal large protein subdomain movement for all systems, except for oxoG:G, which samples conformations close to the open state. In addition, active site geometry and basepairing of the template base with the incoming nucleotide, reveal distortions and misalignments that range from moderate (i.e., oxoG:A_syn) to extreme (i.e., oxoG_anti/syn:G). These results agree with the available kinetic data for pol X and provide structural insights regarding the mechanism by which this polymerase can accommodate incoming nucleotides opposite oxoG. Our simulations also support the notion that α-helix E is involved both in DNA binding and active site stabilization. Our proposed mechanism by which pol X can preferentially accommodate dATP opposite template oxoG further underscores the role that enzyme dynamics and conformational sampling operate in polymerase fidelity and function.     

Evidence of different G-quadruplex DNA binding with biogenic polyamines probed by electrospray ionization-quadrupole time of flight mass spectrometry,circular dichroism and atomic force microscopy

The binding properties of five G-quadruplex oligonucleotides (humtel24, k-ras32, c-myc22, c-kit1 and c-kit2) with polyamines have been investigated by electrospray ionization-quadrupole time of flight mass spectrometry, circular dichroism, melting temperature, atomic force microscopy (AFM) and molecular simulation. The MS results demonstrated that the polyamines and G-quadruplex DNA can form complexes with high affinity, and one molecule of G-quadruplex DNA can combine several molecules (1–5) of polyamines. The binding affinities of the polyamines to DNA were in the order of spermine > spermidine > putrescine. After binding with polyamines, the conformations of the G-quadruplex DNA were significantly changed, and spermine can induce the configurations of k-ras32 and c-kit1 to deviate from their G-quadruplex structures at high concentrations. In the presence of K⁺, the conformations of G-quadruplex DNA were stabilized, while polyamines can also induced alterations of their configurations. Melting temperature experiments suggested that the T_m of the DNA–polyamine complexes obviously increased both in the absence and presence of K⁺. The AFM results indicated that polyamines can induce aggregation of G-quadruplex DNA. Above results illustrated that the polyamines bound with the phosphate backbone and the base-pairs of G-quadruplex structures. Combining with the molecular simulation, the binding mode of the G-quadruplex DNA and polyamines were discussed. The results obtained would be beneficial for understanding the biological and physiological functions of polyamines and provide useful information for development of antitumor drugs.     

Conformations in solution of the stereoisomeric,peracetylated aldohexose dimethyl acetals and diethyl dithioacetals

The conformations in solution of acyclic carbohydrate derivatives having four contiguous asymmetric centers in all eight diastercoisomeric forms have been studied by ¹H-n.m.r. spectroscopy. The 250-MHz, ¹H-n.m.r. spectra for solutions in chloroform-d of eight penta-O-acetylaldohexose dimethyl acetals, and the corresponding diethyl dithioacetals, furnished a complete set of chemical shifts and proton-proton spin-couplings that are interpreted in terms of conformational compositions at room temperature. The galacto and manno derivatives adopt planar, extended conformations, whereas the other six stereoisomers all adopt one or more non-extended (“sickle”) conformations. The results are interpreted on the basis of the avoidance of parallel 1,3-interactions of substituents. The conformational assignments are correlated with observations made previously for aldopentose analogs. An assessment is made of the extent to which valid conformational predictions may be advanced for four-center, and longer, asymmetrically substituted chains, based on observations made for shorter-chain analogs.     

A comparative study on the interaction of the putative anticancer alkaloids,sanguinarine and chelerythrine,with single- and double-stranded,and heat-denatured DNAs

A detailed investigation on the interaction of two benzophenanthridine alkaloids, sanguinarine (SGR) and chelerythrine (CHL), with the double-stranded (ds), heat-denatured (hd), and single-stranded (ss) DNA was performed by spectroscopy and calorimetry techniques. Binding to the three DNA conformations leads to quenching of fluorescence of SGR and enhancement in the fluorescence of CHL. The binding was cooperative for both of the alkaloids with all the three DNA conformations. The binding constant values of both alkaloids with the ds DNA were in the order of 10⁶ M^?1; binding was weak with hd and much weaker to the ss DNA. The fluorescence emission of the alkaloid molecules bound to the ds and hd DNAs was quenched much less compared to those bound to the ss DNA based on competition with the anionic quencher KI. For both double stranded and heat denatured structures the emission of the bound alkaloid molecules was polarized significantly and strong energy transfer from the DNA bases to the alkaloid molecules occurred. Intercalation of SGR and CHL to ds, hd, and ss DNA was proved from these fluorescence results. Calorimetric studies suggested that the binding to all DNA conformations was both enthalpy and entropy favored. Both the alkaloids preferred double-helical regions for binding, but SGR was a stronger binder than CHL to all the three DNA structures.     

How DNA Polymerase X Preferentially Accommodates Incoming dATP Opposite 8-Oxoguanine on the Template

The modified base 8-oxo-7,8-dihydro-2′-deoxyguanosine (oxoG) is a common DNA adduct produced by the oxidation of DNA by reactive oxygen species. Kinetic data reveal that DNA polymerase X (pol X) from the African swine fever virus incorporates adenine (dATP) opposite to oxoG with higher efficiency than the non-damaged G:C basepair. To help interpret the kinetic data, we perform molecular dynamics simulations of pol X/DNA complexes, in which the template base opposite to the incoming dNTP (dCTP, dATP, dGTP) is oxoG. Our results suggest that pol X accommodates the oxoG_syn:A mispair by sampling closed active conformations that mirror those observed in traditional Watson-Crick complexes. Moreover, for both the oxoG_syn:A and oxoG:C ternary complexes, conformational sampling of the polymerase follows previously described large subdomain movements, local residue motions, and active site reorganization. Interestingly, the oxoG_syn:A system exhibits superior active site geometry in comparison to the oxoG:C system. Simulations for the other mismatch basepair complexes reveal large protein subdomain movement for all systems, except for oxoG:G, which samples conformations close to the open state. In addition, active site geometry and basepairing of the template base with the incoming nucleotide, reveal distortions and misalignments that range from moderate (i.e., oxoG:A_syn) to extreme (i.e., oxoG_anti/syn:G). These results agree with the available kinetic data for pol X and provide structural insights regarding the mechanism by which this polymerase can accommodate incoming nucleotides opposite oxoG. Our simulations also support the notion that α-helix E is involved both in DNA binding and active site stabilization. Our proposed mechanism by which pol X can preferentially accommodate dATP opposite template oxoG further underscores the role that enzyme dynamics and conformational sampling operate in polymerase fidelity and function.     

The influence of rotor speed on the sedimentation behavior in sucrose gradients of high molecular weight DNA's

Anomalous sedimentation behavior has been observed for high molecular weight duplex DNA's in sucrose gradients. The sedimentation rate of DNA's having molecular weights of 10⁸ or higher is influenced by high centrifugal fields. The change in the sucrose sedimentation coefficient due to this effect, S^RPM_suc-S⁰_suc, is equal to 1 × 10^?48M^3.65( The anomalous behavior is not influenced by DNA concentration at sufficiently low concentrations. Because of the smallness of the coefficient this effect has not been previously detected for DNA's the size of T2 or smaller at rotor speeds below 40000 RPM. For example, the relative sedimentation coefficient of T2 DNA at 65 000 RPM is only 9% less than at 10000 RPM. However, the sedimentation profile of heterogeneous high molecular weight [(100 – 350) × 10⁶] E. coli DNA is severely altered even at moderate rotor speeds (37000 RPM). Therefore, it seems advisable to use low rotor speeds when sedimenting high molecular weight DNA's.     

Origin of the regioselectivity in an intramolecular nucleophilic addition to arene chromium tricarbonyl complexes

The source of the regioselectivity in the intramolecular nucleophilic addition of nitrile-stabilized carbanions to arene chromium tricarbonyl complexes was investigated for seven different substitution patterns on the arenes. All of the arenes are 1,4-dioxygenated and the substitution varies in the oxygen substituent and in the substituents of the arene carbons (hydrogen and alkyl). The regioselectivity is correlated with the preferred conformations of the chromium tricarbonyl group which in turn was determined by solution and solid-state ¹³C NMR spectroscopy, ¹H NMR spectroscopy in solution as well as X-ray diffraction. In the four complexes analyzed by X-ray diffraction and the three complexes analyzed by solid-state ¹³C NMR spectroscopy, there was only one complex where it was found that the preferred conformation of the -Cr(CO)₃ is different in solution than it is in the solid-state.     

Indirect readout in drug-DNA recognition: role of sequence-dependent DNA conformation

DNA-binding drugs have numerous applications in the engineered gene regulation. However, the drug-DNA recognition mechanism is poorly understood. Drugs can recognize specific DNA sequences not only through direct contacts but also indirectly through sequence-dependent conformation, in a similar manner to the indirect readout mechanism in protein-DNA recognition. We used a knowledge-based technique that takes advantage of known DNA structures to evaluate the conformational energies. We built a dataset of non-redundant free B-DNA crystal structures to calculate the distributions of adjacent base-step and base-pair conformations, and estimated the effective harmonic potentials of mean force (PMF). These PMFs were used to calculate the conformational energy of drug-DNA complexes, and the Z-score as a measure of the binding specificity. Comparing the Z-scores for drug-DNA complexes with those for free DNA structures with the same sequence, we observed that in several cases the Z-scores became more negative upon drug binding. Furthermore, the specificity is position-dependent within the drug-bound region of DNA. These results suggest that DNA conformation plays an important role in the drug-DNA recognition. The presented method provides a tool for the analysis of drug-DNA recognition and can facilitate the development of drugs for targeting a specific DNA sequence.     

Multiprotein E. coli SSB–ssDNA complex shows both stable binding and rapid dissociation due to interprotein interactions

Escherichia coli SSB (EcSSB) is a model single-stranded DNA (ssDNA) binding protein critical in genome maintenance. EcSSB forms homotetramers that wrap ssDNA in multiple conformations to facilitate DNA replication and repair. Here we measure the binding and wrapping of many EcSSB proteins to a single long ssDNA substrate held at fixed tensions. We show EcSSB binds in a biphasic manner, where initial wrapping events are followed by unwrapping events as ssDNA-bound protein density passes critical saturation and high free protein concentration increases the fraction of EcSSBs in less-wrapped conformations. By destabilizing EcSSB wrapping through increased substrate tension, decreased substrate length, and protein mutation, we also directly observe an unstable bound but unwrapped state in which ∼8 nucleotides of ssDNA are bound by a single domain, which could act as a transition state through which rapid reorganization of the EcSSB–ssDNA complex occurs. When ssDNA is over-saturated, stimulated dissociation rapidly removes excess EcSSB, leaving an array of stably-wrapped complexes. These results provide a mechanism through which otherwise stably bound and wrapped EcSSB tetramers are rapidly removed from ssDNA to allow for DNA maintenance and replication functions, while still fully protecting ssDNA over a wide range of protein concentrations.     

A sequence of the yeast 2 micron DNA plasmid chromosome near the origin of replication is exposed to restriction endonuclease digestion

The yeast 2 μm DNA plasmid nucleoprotein complex was subjected to restriction endonuclease digestion to ascertain whether all possible sites are equally accessible to hydrolysis. When plasmid nucleoprotein complexes which had been fixed with formaldehyde were exhaustively digested with restriction endonucleases HinfI or CfoI, only a few of the limit digest products were produced. Furthermore, the limited set of restriction endonuclease sites exposed in formaldehyde-treated plasmid chromosomes could be shown to be preferentially hydrolyzed when plasmid chromosomes which had not been treated with formaldehyde were digested with the same restriction endonucleases. Mapping of the preferred sites revealed that they mapped to the region of the plasmid near the replication origin. These results demonstrate that the protection of DNA from nuclease activity is not constant along the plasmid chromatin, and that a region near the replication origin is preferentially exposed to endonuclease hydrolysis.     

Role of ATP in the cleavage mechanism of the EcoP15 restriction endonuclease

The EcoP15 restriction endonuclease forms complexes at specific sites on unmodified DNA both in the presence and in the absence of S-adenosyl-l-methionine. ATP acts as an allosteric effector of EcoP15 and induces DNA cleavage followed by release of the enzyme from the DNA. The efficiency of endonucleolytic scission varies from site to site. The nucleotide sequences at sites that are cleaved at a high frequency were compared.     

RecO Protein Initiates DNA Recombination and Strand Annealing through Two Alternative DNA Binding Mechanisms

Recombination mediator proteins (RMPs) are important for genome stability in all organisms. Several RMPs support two alternative reactions: initiation of homologous recombination and DNA annealing. We examined mechanisms of RMPs in both reactions with Mycobacterium smegmatis RecO (MsRecO) and demonstrated that MsRecO interacts with ssDNA by two distinct mechanisms. Zinc stimulates MsRecO binding to ssDNA during annealing, whereas the recombination function is zinc-independent and is regulated by interaction with MsRecR. Thus, different structural motifs or conformations of MsRecO are responsible for interaction with ssDNA during annealing and recombination. Neither annealing nor recombinase loading depends on MsRecO interaction with the conserved C-terminal tail of single-stranded (ss) DNA-binding protein (SSB), which is known to bind Escherichia coli RecO. However, similarly to E. coli proteins, MsRecO and MsRecOR do not dismiss SSB from ssDNA, suggesting that RMPs form a complex with SSB-ssDNA even in the absence of binding to the major protein interaction motif. We propose that alternative conformations of such complexes define the mechanism by which RMPs initiate the repair of stalled replication and support two different functions during recombinational repair of DNA breaks.