Proton concentration (pH) switches the higher-order structure of DNA in the presence of spermine

Single-chain observations on the conformational change of giant DNA (T4 DNA) molecules were performed using fluorescence microscopy at different values of pH in the presence of spermine. Individual DNA molecules undergo a large discrete change, or all-or-none transition, in conformation from a folded compact state to an unfolded coil state with an increase in pH. This abrupt unfolding of DNA with an increase in pH is attributed to a decrease in the concentration of the tetravalent form in spermine [SPM(4+)]. We propose a scheme for the folding transition of single DNAs, where the manner of spermine binding changes dramatically from weak loose binding in the elongated coil state to strong tight binding in the folded compact state. We discuss the hierarchical nature of the transition, i.e. cooperative continuous change on the ensemble vs. all-or-none switching on individual DNAs.     

Alcohol induced B-A transition of DNAs with different base compositions studied by circular dichroism

The circular dichroic (CD) spectra of natural DNAs (from Cl. perfringens, T2 phage, calf thymus, E. coli, and M. lysodeikticus) as well as duplexes of synthetic DNAs (poly(dA) X poly(dT), poly(dA-dT), and poly(dG-dC] were measured in water-ethanol mixtures with 0.3 mM NaCl. A conformational change from the B to the A form was observed for the natural DNAs on adding ethanol. The ethanol concentration that induces the transition and the extent of the change in the CD spectrum are different for the five natural DNAs depending on their GC contents. The higher the GC content is, the more easily the transition to the A form takes place. The results indicate that the GC content of a DNA is an important factor for induction of the B-A transition. The results for the synthetic DNAs show that their properties cannot be inferred by simple extrapolation of those of natural DNAs. Coexisting ions and the molecular weight of a DNA were also found to affect the induction of the B-A transition.     

The B-A transition in superhelical DNA.

Relaxation of a DNA superhelical stress due to the B to A transition induced by trifluoroethanol has been studied by assessing the change of DNA orientation in a flow gradient. Using DNAs of different superhelical densities, a decrease in the winding angle during the B----A shift of DNA was found to be 1.5 degrees per base pair in solution. Accepting the winding angle for B-DNA in solution to be 34.1 degrees, that for A-DNA must have a value of 32.6 degrees which agrees with the X-ray data for A-DNA in the condensed state. The date obtained within the B-A transition interval make it possible to conclude that there is an increase in winding at each B/A junction, which is about 5 degrees per one junction.     

The helix-coil transition of closed and nicked DNAs in aqueous neutral trichloroacetate solutions.

The melting transition for closed, underwound DNAs and for nicked or linear DNAs was monitored by velocity sedimentation and by absorbance spectroscopy in aqueous NaCCl3CO2 (NaTCA) and RbTCA. The addition of neutral trichloroacetate lowers the midpoint of the helix-coil transition by 26% C/M for RbTCA and by 32% C/M for NaTCA, depressing the denaturation region to near room temperature at neutral pH. The melting of nicked DNA is cooperative, occurring over a temperature range of about 5.6 degrees C. The melting profile for closed DNA is broad and noncooperative with a transition breadth greater than 45 degrees. Closed DNAs undergo a structural alteration, as revealed by velocity sedimentation, resulting in a reduction in the number of superhelical turns at temperatures and salt concentrations substantially below the melting temperatures and salt concentrations substantially below the melting temperature of the nicked DNA. The reduction in the extent of supercoiling continues upon isothermal addition of salt up to the salt concentration at which all superhelical turns are removed. The salt concentration at the principal minimum in the sedimentation velocity profile (3.16 M NaTCA for PM-2 DNA) is approximately the same as that at the midpoint of the helix-coil transition for the nicked DNA.     

Disappearance of the negative charge in giant DNA with a folding transition

In the present study we measure the electrophoretic mobility of giant T4 DNA (166 kbp) by electrophoretic light scattering for the elongated and folded compact states at different spermidine (trivalent cation) concentrations in 50 mM sodium maleate buffer (pH 6.0). It is found that the electrophoretic mobility of elongated DNA in the absence of the multivalent cation is seven times greater than that of fully folded compact DNA, where, with the increase of the concentration of spermidine, an abrupt transition is generated after a gradual decrease of the mobility. An analysis of the electrophoretic mobility suggests that the folded compact DNA chains almost completely lose their negative charges, by taking into account the difference of friction mechanism between an elongated and folded compact state. From the single chain observation by use of fluorescence microscopy, it is found that a phase-segregated structure is generated at intermediate concentrations of spermidine. The gradual decrease of the electrophoretic mobility in the transition region is, thus, attributed to the formation of the segregated state, exhibiting partial electroneutralization in the folded part. Disappearance of the negative charges in the completely folded compact DNAs is discussed in relation to the mechanism of transition, in terms of a first-order phase transition.     

Circular dichroism studies of the B goes to A conformational transition in seven small DNA restriction fragments containing the Escherichia coli lactose control region.

The B goes to A conformational transition caused by high ethanol concentrations was studied for seven DNA restriction fragments with overlapping and known sequences. Since the DNAs are homogeneous and range in GC content from 44-63%, they permit an evaluation of the influence of DNA sequence and base composition on the B goes to A transition. Moreover, their small size (80-301 bp) minimizes precipitation artifacts. The B- form spectra (in low salt) and the transition toward the C- form (in ethanol concentrations below the B goes to A transition) agree with prior measurements on chromosomal DNAs and are similar for all seven DNAs. At higher ethanol concentrations (80%), all fragments undergo a transition to the A- form as judged by the large increase of the positive CD band at 270 nm. Difference spectra among the fragments reveal minor differences between the A- form spectra. The ethanol concentration necessary to cause this transition is 72 +/- 2% for all fragments, thus excluding a preference of the CAP-, E. coli RNA polymerase-, or lac repressor-binding sequences for the A- form. The kinetics of the B goes to A transition in 80% ethanol are biphasic; the initial rapid transition is an intramolecular B goes to A form shift and the slower transition is an aggregation (but not precipitation) of the DNA     

A DNA pairing-enhanced conformation of bacterial RecA proteins

The RecA proteins of Escherichia coli (Ec) and Deinococcus radiodurans (Dr) both promote a DNA strand exchange reaction involving two duplex DNAs. The four-strand exchange reaction promoted by the DrRecA protein is similar to that promoted by EcRecA, except that key parts of the reaction are inhibited by Ec single-stranded DNA-binding protein (SSB). In the absence of SSB, the initiation of strand exchange is greatly enhanced by dsDNA-ssDNA junctions at the ends of DNA gaps. This same trend is seen with the EcRecA protein. The results lead to an expansion of published hypotheses for the pathway for RecA-mediated DNA pairing, in which the slow first order step (observed in several studies) involves a structural transition to a state we designate P. The P state is identical to the state found when RecA is bound to double-stranded (ds) DNA. The structural state present when the RecA protein is bound to single-stranded (ss) DNA is designated A. The DNA pairing model in turn facilitates an articulation of three additional conclusions arising from the present work. 1) When a segment of a RecA filament bound to ssDNA is forced into the P state (as RecA bound to the ssDNA immediately adjacent to dsDNA-ssDNA junction), the segment becomes "pairing enhanced." 2) The unusual DNA pairing properties of the D. radiodurans RecA protein can be explained by postulating this protein has a more stringent requirement to initiate DNA strand exchange from the P state. 3) RecA filaments bound to dsDNA (P state) have directly observable structural changes relative to RecA filaments bound to ssDNA (A state), involving the C-terminal domain.     

Free energy landscape and transition pathways from Watson–Crick to Hoogsteen base pairing in free duplex DNA

Houghton (HG) base pairing plays a central role in the DNA binding of proteins and small ligands. Probing detailed transition mechanism from Watson–Crick (WC) to HG base pair (bp) formation in duplex DNAs is of fundamental importance in terms of revealing intrinsic functions of double helical DNAs beyond their sequence determined functions. We investigated a free energy landscape of a free B-DNA with an adenosine–thymine (A–T) rich sequence to probe its conformational transition pathways from WC to HG base pairing. The free energy landscape was computed with a state-of-art two-dimensional umbrella molecular dynamics simulation at the all-atom level. The present simulation showed that in an isolated duplex DNA, the spontaneous transition from WC to HG bp takes place via multiple pathways. Notably, base flipping into the major and minor grooves was found to play an important role in forming these multiple transition pathways. This finding suggests that naked B-DNA under normal conditions has an inherent ability to form HG bps via spontaneous base opening events.     

Precollapse of T7 DNA by spermidine at low ionic strength: A linear dichroism and intrinsic viscosity study

At low salt ([Na⁺] = 10^?3M), spermidine is capable of transforming DNA from a highly extended random coil to a compact particle. The transition takes place at a spermidine concentration of around 25 μM and the compact particle has been previously studied in considerable detail for several different DNAs. The objective of the present study is to see what effect, if any, spermidine has on T7 DNA conformation prior to collapse using flow dichroism and intrinsic viscosity. We conclude that increasing the spermidine concentration from 0 to the collapse transition point (above 20 μM) makes DNA increasingly nondraining. Furthermore, the persistence length dropped from 785 (±42) to 560 (±32) to 445 (±26) Å on increasing the ambient spermidine concentration from 0 to 1 to 10 μM. These results are in good agreement with counterion condensation theory and Odijk's theory of the electrostatic contribution to the persistence length of DNA. Nonetheless, it is concluded that counterion condensation is not entirely responsible for DNA collapse and that crosslinking promotes the transition to the compact state.     

Characterization of imidazole as a DNA denaturant by using TGGE of PCR products from a random pool of DNA

Perpendicular temperature gradient gel electrophoresis (TGGE) profiles were analyzed for PCR products from a random pool of DNA [60 nts random region flanked by two primer (20 nts) sites]. Besides a normal transition profile of a homoduplex, unique mobility transition profiles of two kinds of heteroduplex with a big internal loop were observed, representing the successive helix-coil transitions of the DNAs. As the appearance of the heteroduplex band is an estimator of the complexity of a random pool, it will be applicable to monitor the extent of the selection process in the in vitro selection method. When imidazole was added to the electrophoretic buffer, the transition pattern shifted to the low temperature side. At a concentration of 1 M, imidazole lowered the melting temperature (Tm) of DNA by 13+/-2 degrees C for all the three chain separation transitions observed. Thus imidazole is a stronger denaturant than urea, at least at dilute concentration. Dependence of Tm on concentration of imidazole and the mobility change suggested that imidazole binds to nucleotide in the single-stranded state.     

Sea urchin DNA methyltransferases

DNA methyltransferase activities have been partially purified from unfertilized eggs and blastula nuclei of sea urchin embryos. Comparative studies, using different DNAs as substrates, show that the two preparations are most active on hemimethylated and single-strand DNA, but they methylate, though at a lower rate, also on double-strand DNA. The two activities show distinctive efficiencies in methylating plasmid DNAs and marked differences in the rate of methyl transfer to DNAs in different structural states: linear, relaxed, or supercoiled. The ratio of the apparent specific activity of the two preparations depends on the particular DNA used as substrate and its structure. Methylation analysis of the restriction fragments of methylated plasmid DNAs shows a linear correlation between introduced methyl groups and the percent of CpG of each particular fragment, indicating that methylation is substantially random and sequence is less relevant than conformation in determining enzyme efficiency. The data do not permit us to decide if the two activities are different enzymes or the same enzyme with different modulating factors.     

Folding and unfolding of a giant duplex-DNA in a mixed solution with polycations, polyanions and crowding neutral polymers

To understand the conformational behavior of a giant duplex-DNA chain in a mixed solution with various biopolymers with different state of ionization, the higher-order structure of the DNA chain was analyzed with a fluorescence microscope in the presence of polycations (poly-arginine), polyanions (poly-glutamic acid), and neutral polymers (poly-ethylene glycol) as a model for cellular environment. Concentrated medium with neutral polymer induced the discrete folding transition of the DNA. At the threshold condition for the transition, addition of small amounts of either the polycation or the polyanion caused marked structural changes in the folded DNAs. Based on thermodynamic considerations on the experimental results, profile of free energy of a single giant DNA chain was depicted with respect to the size, or the expansion factor alpha, in the three-dimensional structure of the DNA. The effect of the neural crowding polymer on the degree of folding of a single giant DNA chain is discussed in a semi-quantitative manner.     

Comparison of plasmid DNA topology among mesophilic and thermophilic eubacteria and archaebacteria.

Several plasmid DNAs have been isolated from mesophilic and thermophilic archaebacteria. Their superhelical densities were estimated at their host strain's optimal growth temperature, and in some representative strains, the presence of reverse gyrase activity (positive DNA supercoiling) was investigated. We show here that these plasmids can be grouped in two clusters with respect to their topological state. The group I plasmids have a highly negatively supercoiled DNA and belong to the mesophilic archaebacteria and all types of eubacteria. The group II plasmids have DNA which is close to the relaxed state and belong exclusively to the thermophilic archaebacteria. All archaebacteria containing a relaxed plasmid, with the exception of the moderately thermophilic methanogen Methanobacterium thermoautotrophicum Marburg, also exhibit reverse gyrase activity. These findings show that extrachromosomal DNAs with very different topological states coexist in the archaebacterial domain.     

Oligomerization of a MutS mismatch repair protein from Thermus aquaticus.

The MutS DNA mismatch protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. We have examined the oligomerization of a MutS protein from Thermus aquaticus that binds to heteroduplex DNAs at elevated temperatures. Analytical gel filtration, cross-linking of MutS protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq protein is largely a dimer in free solution. Analytical equilibrium sedimentation showed that the oligomerization of Taq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 microM. The DeltaG(0)(2-4) for the dimer to tetramer transition is approximately -6.9 +/- 0.1 kcal/mol of tetramer. Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding protein-MutS fusion protein bound to a short, 37-base pair heteroduplex DNA reveal that the protein binds to DNA as a dimer with no change in oligomerization upon DNA binding.     

DNA B to D transition can be explained in terms of hydration economy of the minor groove atoms

Adjacent phosphate oxygen atoms in A and Z-DNA are located much closer together than in the B form and can be hydrated more economically due to the formation of water bridges between them, whereas in the B form phosphates are hydrated individually. This principle of hydration economy of phosphate groups discovered by Saenger and colleagues could not be applied to the B-D transition, which, like the B-A and B-Z transitions, occurs in a situation of water deficiency, because the distances between adjacent phosphates of individual polynucleotide chains in the D form are not much different from B-DNA. It follows from our calculations of B and D-DNA accessibility to solvent performed by the method of Lee & Richards, and from a simulation of solvent structure near DNA, that there is an economy of hydration only for the minor groove atoms. This feature and some experimental data can explain why only a limited range of sequences consisting of A.T or I.C pairs undergo the transition to the D form. The conformational transition in DNAs with such sequences to a poly[d(A]).poly[d(T])-like conformation (Bh-DNA), which is accompanied by a narrowing of the minor groove, can be explained in the same way. Calculations suggest that in the D-form minor groove of different A-T or I-C DNAs there is a double-layer hydration spine similar to that observed by Drew & Dickerson in the A-T tract of the d(C-G-C-G-A-A-T-T-C-G-C-G) dodecamer. The B-D and B-Bh transitions in A + T-rich DNAs can have biological implications, e.g. they can facilitate DNA bending upon the interaction with proteins.     

Replication of mammalian DNA in bromodeoxyuridine: Appearance of a component with intermediate density

Summary When three lines of mammalian cells were cultured with 5-bromodeoxyuridine (BrdUrd) for less than one generation, their DNAs displayed three peaks in CsCl gradients. In addition to the expected unsubstituted (LL) and hybrid (LH) peaks, there was a significant absorbance peak of intermediate density (INT) between LH and LL DNAs. This INT DNA has characteristics expected of an intermediate of DNA replication. Upon shearing, it behaves as though it contains contiguous segments of unsubstituted and hybrid DNAs. Upon continuous exposure of cells to [³H]-BrdUrd, radioactivity accumulates in INT DNA for 60–90 min when a steady state condition is reached. At that time, the rate of incorporation into LH DNA increases, consistent with a precursorproduct relationship. In a pulse-chase experiment, radioactivity is chased from INT DNA into LH DNA. To account for the above observations and for the size and sharpness of the INT DNA peak in CsCl, we suggest that a high molecular weight replication intermediate accumulates before completing replication into mature daughter molecules.     

不同生理状态下膜状急纤虫大核DNA和线粒体DNA的多态性比较

为探索纤毛虫在营养及休眠条件下两套遗传系统的作用关系,对膜状急纤虫(Tachysomapellionella)营养细胞和休眠包囊大核DNA、线粒体DNA进行了RAPD比较。结果显示,在所选用的34条随机引物中,大核DNA共扩增出203条片段,其中以休眠包囊大核DNA为模板扩增出45条特有片段,以营养细胞大核DNA为模板扩增出36条特有片段,两者存在40%的差异。在所选用的32条随机引物中,线粒体DNA共扩增出216条片段,其中以休眠包囊线粒体DNA为模板扩增出35条特有片段,以营养细胞线粒体DNA为模板扩增出47条特有片段,两者有38%的差异。结果表明,膜状急纤虫休眠包囊与营养期的大核DNA结构存在显著的差异;两者的线粒体DNA结构也存在较大差异。这表明,膜状急纤虫在包囊形成过程中,大核及线粒体DNA结构可能都发生了一定的变化,并且这些变化可能与包囊形成过程中的形态结构和代谢活动等剧烈变化以及休眠状态下的生理生化变化密切相关。