The central helix of calmodulin functions as a flexible tether

Using site-directed mutagenesis we have created an altered calmodulin in which Gln-3 and Thr-146 have both been replaced by cysteines. We have reacted this protein with the bifunctional reagent, bismaleimidohexane, forming an intramolecular cross-link between the two cysteines. In the crystal structure of native calmodulin alpha-carbons at positions 3 and 146 are 37 A apart. In the bismaleimidohexane cross-linked protein these atoms can be no more than 19 A apart, and model building studies indicate that there is probably a bend in the central helix of calmodulin. A second modified calmodulin was generated by cleaving the central helix of the cross-linked protein at Lys-77 with trypsin. In this molecule, the two lobes of calmodulin are joined solely by the bismaleimidohexane cross-link, which bridges Cys-3 and Cys-146. Vm and Kact values for activation of myosin light chain kinase activity by the cross-linked and cross-linked/trypsinized proteins are not significantly different from those for the control protein. This result indicates that one role for the central helix may be to serve as a flexible tether between the calmodulin lobes. This is consistent with a model calmodulin-enzyme complex in which the central helix is bent, and the two lobes exert a concerted effect. A detailed model of this type has been proposed for the calmodulin-myosin light chain kinase complex (Persechini, A. and Kretsinger, R.H. (1988) J. Cardiovasc. Pharmacol., in press).     

Strong bending of the DNA double helix

During the past decade, the issue of strong bending of the double helix has attracted a lot of attention. Here, we overview the major experimental and theoretical developments in the field sorting out reliably established facts from speculations and unsubstantiated claims. Theoretical analysis shows that sharp bends or kinks have to facilitate strong bending of the double helix. It remains to be determined what is the critical curvature of DNA that prompts the appearance of the kinks. Different experimental and computational approaches to the problem are analyzed. We conclude that there is no reliable evidence that any anomalous behavior of the double helix happens when DNA fragments in the range of 100 bp are circularized without torsional stress. The anomaly starts at the fragment length of about 70 bp when sharp bends or kinks emerge in essentially every molecule. Experimental data and theoretical analysis suggest that kinks may represent openings of isolated base pairs, which had been experimentally detected in linear DNA molecules. The calculation suggests that although the probability of these openings in unstressed DNA is close to 10⁻⁵, it increases sharply in small DNA circles reaching 1 open bp per circle of 70 bp.     

Tau could protect DNA double helix structure

The hyperchromic effect has been used to detect the effect of tau on the transition of double-stranded DNA to single-stranded DNA. It was shown that tau increased the melting temperature of calf thymus DNA from 67 to 81 degrees C and that of plasmid from 75 to 85 degrees C. Kinetically, rates of increase in absorbance at 260 nm of DNA incubated with tau were markedly slower than those of DNA and DNA/bovine serum albumin used as controls during thermal denaturation. In contrast, rates of decrease in the DNA absorbance with tau were faster than those of controls when samples were immediately transferred from thermal conditions to room temperature. It revealed that tau prevented DNA from thermal denaturation, and improved renaturation of DNA. Circular dichroic spectra results indicated that there were little detectable conformational changes in DNA double helix when tau was added. Furthermore, tau showed its ability to protect DNA from hydroxyl radical (.OH) attacking in vitro, implying that tau functions as a DNA-protecting molecule to the radical.     

Nonhistone proteins HMG1 and HMG2 unwind DNA double helix.

In a previous communication we have shown that both HMG1 and HMG2 nonhistone proteins change the DNA helical structure and the binding of HMG1 and HMG2 to DNA induces a net unwinding equivalent of DNA double helix (Javaherian, K., Liu, L. F. and Wang, J. C. (1978) Science, 199, 1345-1346). Employing melting absorption technique, we now show that in the presence of salt HMG1 and HMG2 destabilize DNA whereas in the absence of salt, they both stabilize DNA molecules. Consequently the folded structure of HMG must play an important role in melting DNA. Furthermore, by measuring topological winding number using competition unwinding experiments, we conclude that HMG1 has a higher affinity for a single-stranded DNA relative to double-stranded DNA. These results together suggest that HMG1 and HMG2 unwind DNA double helix by local denaturation of the DNA base pairs. The net unwinding angles have been measured to be 22 degrees and 26 degrees per molecule of HMG1 and HMG2 respectively.     

Sequence-dependent base pair opening in DNA double helix

Preservation of genetic information in DNA relies on shielding the nucleobases from damage within the double helix. Thermal fluctuations lead to infrequent events of the Watson-Crick basepair opening, or DNA "breathing", thus making normally buried groups available for modification and interaction with proteins. Fluctuational basepair opening implies the disruption of hydrogen bonds between the complementary bases and flipping of the base out of the helical stack. Prediction of sequence-dependent basepair opening probabilities in DNA is based on separation of the two major contributions to the stability of the double helix: lateral pairing between the complementary bases and stacking of the pairs along the helical axis. The partition function calculates the basepair opening probability at every position based on the loss of two stacking interactions and one base-pairing. Our model also includes a term accounting for the unfavorable positioning of the exposed base, which proceeds through a formation of a highly constrained small loop, or a ring. Quantitatively, the ring factor is found as an adjustable parameter from the comparison of the theoretical basepair opening probabilities and the experimental data on short DNA duplexes measured by NMR spectroscopy. We find that these thermodynamic parameters suggest nonobvious sequence dependent basepair opening probabilities.     

Force-induced melting of the DNA double helix 1. Thermodynamic analysis

The highly cooperative elongation of a single B-DNA molecule to almost twice its contour length upon application of a stretching force is interpreted as force-induced DNA melting. This interpretation is based on the similarity between experimental and calculated stretching profiles, when the force-dependent free energy of melting is obtained directly from the experimental force versus extension curves of double- and single-stranded DNA. The high cooperativity of the overstretching transition is consistent with a melting interpretation. The ability of nicked DNA to withstand forces greater than that at the transition midpoint is explained as a result of the one-dimensional nature of the melting transition, which leads to alternating zones of melted and unmelted DNA even substantially above the melting midpoint. We discuss the relationship between force-induced melting and the B-to-S transition suggested by other authors. The recently measured effect on T7 DNA polymerase activity of the force applied to a ssDNA template is interpreted in terms of preferential stabilization of dsDNA by weak forces approximately equal to 7 pN.     

The effect of the superhelicity on the double helix twist angle in DNA.

An attempt to estimate the relative contributions of twisting and bending to the free energy of superhelix formation from the relaxed DNA is undertaken. The extent of teritiary ordering (number of DNA axis turns tau) and that of secondary ordering (duplex twist angle beta) have been taken as thermodynamical parameters, which characterize the state of the supercoild DNA at the fixed linking number (Lk) value. Such a thermodynamical approach implies the phenomenological parameters of rigidities of twisting and supercoiling (Gbeta, Gtau). Gtau/Gbeta ratio is estimated from the presented experimental data on the winding of the double helix upon increasing the ionic strength when twist alterations are followed by circular dichroism method. The adequacy of such interpretation of CD spectra changes are discussed. The values of Gtau and Gbeta are estimated to be of the same order of magnitude.     

Complex between triple helix of collagen and double helix of DNA in aqueous solution

We demonstrate in this paper that one example of a biologically important and molecular self-assembling complex system is a collagen–DNA ordered aggregate which spontaneously forms in aqueous solutions. Interaction between the collagen and the DNA leads to destruction of the hydration shell of the triple helix and stabilization of the double helix structure. From a molecular biology point of view this nano-scale self-assembling superstructure could increase the stability of DNA against the nucleases during collagen diseases and the growth of collagen fibrills in the presence of DNA.     

Interaction between double helix DNA fragments and a new topopyrone acting as human topoisomerase I poison

A water soluble derivative (2) of topopyrones was selected for NMR studies directed to elucidate the mode of binding with specific oligonucleotides. Topopyrone 2 can intercalate into the CG base pairs, but the residence time into the double helix is very short and a fast chemical exchange averaging occurs at room temperature between the free and bound species. The equilibria involved become slow below room temperature, thus allowing to measure a mean lifetime of the complex of ca. 7 ms at 15 °C. Structural models of the complex with d(CGTACG)₂ were developed on the basis of DOSY, 2D NOESY and ³¹P NMR experiments. Topopyrone 2 presents a strong tendency to self-associate. In the presence of oligonucleotide a certain number of ligand molecules are found to externally stack to the double-helix, in addition to a small fraction of the same ligand intercalated. The external binding to the ionic surface of the phosphoribose chains may thus represents the first step of the intercalation process.     

Hydrogen-bonding effects and 13C-NMR of the DNA double helix

13C-nmr chemical shifts of the nucleotides in DNA are sensitive to hydrogen bonding, especially for three of the carbons immediately bonded to exocyclic oxygen or nitrogen atoms acting as H-bond acceptors or donors. GuoC2, GuoC6 and ThdC4 are strongly deshielded (about 1 ppm) upon Watson-Crick pairing in oligodeoxynucleotide duplexes, regardless of the base sequence. Deshielding at these sites may be useful to distinguish bases involved in Watson-Crick pairs from unpaired bases.     

Interactions of caffeine with DNA double helix fragments. Molecular mechanics simulation

Calculations of the energy of interaction between the caffeine molecule and DNA double helix fragment of four complementary pairs have been performed by the molecular mechanics method. The calculations demonstrate the existence of energy minima corresponding to the caffeine molecule position in both wide and narrow grooves. Each of three proton acceptor atoms of caffeine is able to form hydrogen bond with each of three amino groups of DNA bases. The interactions of caffeine with both hydrogen bonded nucleotide and other nucleotides of the two strands contribute considerably to the total energy. The substantial contribution of interactions of caffeine with other than H-bonded nucleotides results in a rather close packing of atom groups in possible DNA-caffeine complexes. The mechanisms of influence of caffeine on interactions of DNA with other biologically active compounds are discussed.     

Statistical-mechanical treatment of violations of the double helix in supercoiled DNA.

We consider the problem of making allowance for superhelicity in the statistical-mechanical calculations of fluctuational violations of the DNA double helix. A simple model is discussed, making it possible in the calculations to use an approach based on the theory of helix–coil transition in DNA. The proposed algorithms allow calculating the effect of superhelicity on the base-pair fluctuational opening for any given sequence of nucleotides. An algorithm is also proposed allowing for the hairpin and cruciform structures in the palindromic regions of a sequence, as well as the open and helical states. The theory is used to calculate the melting curve for superhelical DNA at temperatures well below the melting point of the linear or nicked forms. The maps of opening probability are calculated for SV40 and ?X174 DNA using their recently published complete nucleotide sequences. The data explain well the experimental results of probing the secondary structure of these DNA by single strand-specific endonucleases.     

Resonant and localized breathing modes in terminal regions of the DNA double helix.

A Green's function approach is used in constructing a dynamic model of a semi-infinite length of the DNA homopolymer B poly(d) . poly(d). Considerable attention is focused on the hydrogen bond stretching close to the terminus. A melting (or breathing) coordinate (M) is defined as an average over the three linking hydrogen bond stretches in a unit cell. The thermal mean squared amplitude of (M) is enhanced at the chain end compared with the interior. Spectral branches at 69, 80 and 105 cm-1, as well as a local mode at 75 cm-1, are primary contributors to the enhancement. We suggest that this fact can affect the thermal melting of a DNA double helical homopolymer, enhancing the tendency to start from an end (if one is available). We show how certain infinite chain modes with small (M) amplitude can turn into breathing modes near the terminus, and suggest that the same phenomenon may occur near other specific base-pair sequences. There is also considerable attention paid to the low microwave region from approximately 0 to 1.75 cm-1. The thermally activated modes in this frequency region contribute approximately (0.02 A)2 to [M2(0)] at 40 K, approximately two orders of magnitude greater than for [M2(infinity)]. Most important however, is the existence of narrow resonant modes in this frequency region. Particularly pronounced resonances near 0.03 cm-1 and 0.08 cm-1 (approximately 0.9 and 2.4 GHz) amplify M2(0) at the terminus by about for orders of magnitude over the infinite chain value M2(infinity).