Experimental method for studying conformational transitions in DNA

An experimental method combining fiber X-ray and direct fiber dimension measurements is proposed for the study of DNA conformational transitions. Curves corresponding to the A-B and B-C transitions are obtained by using the proportionality which exists between the fiber length and the axial rise per nucleotide in the DNA helix. The A-B transition is shown to be cooperative while the B-C one is a progressive change of helical conformation.     

Changes of hydration during conformational transitions of DNA

Fiber X-ray diffraction and measurement of fiber dimensions yields information about the hydration of DNA in fibers. The results obtained give us the fraction of nucleotides in the B form for the A-B transition or the rate of progression for the B-C transition as functions of the number of water molecules per nucleotide. The present experimental results confirm the importance of cooperativity in the A-B transition and the progressive change of the DNA double helix conformation during the C-B transition. At least twenty additional water molecules per nucleotide are necessary to stabilize the B form for DNA molecules in fibers following the A to B transition whereas only ten are sufficient when the B conformation is obtained starting from the C form. Offprint requests to: S. Premilat     

Cooperative fluctuations point to the dimerization interface of p53 core domain

Elastic network models are used for investigation of the p53 core domain functional dynamics. Global modes of motion indicate high positive correlations for residue fluctuations across the A-B interface, which are not observed at the B-C interface. Major hinge formation is observed at the A-B interface upon dimerization indicating stability of the A-B dimer. These findings imply A-B as the native dimerization interface, whereas B-C is the crystal interface. The A-B dimer exhibits an opening-closing motion about DNA, supporting the previously suggested clamp-like model of nonspecific DNA binding followed by diffusion. Monomer A has limited positive correlations with DNA, while monomer B exhibits high positive correlations with DNA in the functionally significant slow modes. Thus, monomer B might seem to maintain the stability of the dimer-DNA complex by forming the relatively fixed arm of the dimer clamp, whereas the other arm of the clamp, monomer A, might allow sliding via continuous association/dissociation mechanisms.     

Host range determinants located on the interior of the poliovirus capsid.

The inability of certain poliovirus strains to infect mice can be overcome by the expression of human poliovirus receptors in mice or by the presence of a particular amino acid sequence of the B-C loop of the viral capsid protein VP1. We have identified changes in an additional capsid structure that permit host-restricted poliovirus strains to infect mice. Variants of the mouse-virulent P2/Lansing strain were constructed containing amino acid changes, deletions and insertions in the B-C loop of VP1. These variants were attenuated in mice, demonstrating the importance of the B-C loop sequence in host range. Passage of two of the B-C loop variants in mice led to the selection of viruses that were substantially more virulent. The increased neurovirulence of these strains was mapped to two different suppressor mutations in the N-terminus of VP1. Whereas the B-C loop of VP1 is highly exposed on the surface of the capsid, near the five-fold axis of symmetry, the suppressor mutations are in the interior of the virion, near the three-fold axis. Introduction of the suppressor mutations into the genome of the mouse-avirulent P1/Mahoney strain resulted in neurovirulent viruses, demonstrating that the P2/Lansing B-C loop sequence is not required to infect mice. Because the internal host range determinants are in a structure known to be important in conformational transitions of the virion, the host range of poliovirus may be determined by the ability of virions to undergo transitions catalyzed by cell receptors.     

Conformational changes in DNA and soft modes

In this paper we explore the applicability of the soft mode approach to study the conformational transitions of DNA. It is believed that the A-B conformation change is a first order transition. Soft mode theories only apply to the initial stages of a first order transition. However the mode softening in such a case can be the initiating factor which ultimately leads to the transition. The first order transition is, then, a breakdown of what otherwise would have been a true second order transition. The mode softening is causally connected to onset of the transition. We use the eigenvectors obtained from lattice dynamics calculations to identify the softmode. We use the eigenvector projections to form a force constant matrix that is required to drive a mode soft. We explore the methods by which this force constant matrix can be formed. We suggest that the breaking of specific "water bridges" between phosphate groups in the two single strands can drive the conformation change.     

Solution NMR structure and backbone dynamics of the PsaE subunit of photosystem I from Synechocystis sp. PCC 6803

PsaE is a small peripheral subunit of photosystem I (PSI) that is very accessible to the surrounding medium. It plays an essential role in optimizing the interactions with the soluble electron acceptors of PSI, ferredoxin and flavodoxin. The solution structure of PsaE from the cyanobacterium Synechocystis sp. PCC 6803 has been investigated by NMR with a special emphasis on its protein dynamic properties. PsaE is characterized by a well-defined central core that consists of a five-stranded beta-sheet (+1, +1, +1, -4x). Four loops (designated the A-B, B-C, C-D, and D-E loops) connect these beta-strands, the overall resulting structure being that of an SH3-like domain. As compared to previously determined PsaE structures, conformational differences are observed in the first three loops. The flexibility of the loops was investigated using (15)N relaxation experiments. This flexibility is small in amplitude for the A-B and B-C loops, but is large for the C-D loop, particularly in the region corresponding to the missing sequence of Nostoc sp. PCC 8009. The plasticity of the connecting loops in the free subunit is compared to that when bound to the PSI and discussed in relation to the insertion process and the function(s) of PsaE.     

Conformational Changes in DNA and Soft Modes

Abstract

In this paper we explore the applicability of the soft mode approach to study the conformational transitions of DNA. It is believed that the A-B conformation change is a first order transition. Soft mode theories only apply to the initial stages of a first order transition. However the mode softening in such a case can be the initiating factor which ultimately leads to the transition. The first order transition is, then, a breakdown of what otherwise would have been a true second order transition. The mode softening is causally connected to onset of the transition. We use the eigenvectors obtained from lattice dynamics calculations to identify the softmode. We use the eigenvector projections to form a force constimi matrix that is required to drive a mode soft. We explore the methods by which this force constant matrix can be formed. We suggest that the breaking of specific “water bridges” between phosphate groups in the two single strands can drive the conformation change.     

Water-DNA interactions as studied by X-ray and neutron fibre diffraction

X-ray fibre-diffraction studies indicate a high degree of stereochemical specificity in interactions between water and the DNA double helix. Evidence for this comes from data that show that the molecular conformations assumed by DNA in fibres are highly reproducible and that the hydration-driven transitions between these conformations are fully reversible. These conformational transitions are induced by varying the relative humidity of the fibre environment and hence its water content. Further evidence for stereochemical specificity comes from the observed dependence of the conformation assumed on the ionic content of the fibre and the nucleotide sequence of the DNA. For some transitions, information on stereochemical pathways has come from real-time X-ray fibre diffraction using synchrotron radiation; information on the location of water with respect to the double helix for a number of DNA conformations has come from neutron fibre diffraction. This structural information from fibre-diffraction studies of DNA is complemented by information from X-ray single-crystal studies of oligonucleotides. If the biochemical processes involving DNA have evolved to exploit the structural features observed in DNA fibres and oligonucleotide single crystals, the challenges in developing alternatives to a water environment can be expected to be very severe.     

DNA lesion alters global conformational dynamics of Y-family DNA polymerase during catalysis

A major product of oxidative damage to DNA, 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG), can lead to genomic mutations if it is bypassed unfaithfully by DNA polymerases in vivo. However, our pre-steady-state kinetic studies show that DNA polymerase IV (Dpo4), a prototype Y-family enzyme from Sulfolobus solfataricus, can bypass 8-oxoG both efficiently and faithfully. For the first time, our stopped-flow FRET studies revealed that a DNA polymerase altered its synchronized global conformational dynamics in response to a DNA lesion. Relative to nucleotide incorporation into undamaged DNA, three of the four domains of Dpo4 undertook different conformational transitions during 8-oxoG bypass and the subsequent extension step. Moreover, the rapid translocation of Dpo4 along DNA induced by nucleotide binding was significantly hindered by the interactions between the embedded 8-oxoG and Dpo4 during the extension step. These results unprecedentedly demonstrate that a Y-family DNA polymerase employs different global conformational dynamics when replicating undamaged and damaged DNA.     

The A--B transition: temperature and base composition effects on hydration of DNA

Natural DNAs and some polynucleotides organised in fiber present the A--B form transition at a relative humidity (r.h.) which depends on the temperature. A shift of the midpoint of that helix--helix transition to higher r.h. values is observed when the temperature is risen. It is shown that the average number of water molecules associated to a nucleotide pair is the relevant parameter for the A-B transition and that this parameter can be given a precise value by a combination of different r.h. and temperature values. The minimum number of water molecules necessary to get the B form depends on the base composition of the DNA. It is observed that AT base pairs have a higher affinity toward water molecules than GC base pairs. In the B form there are 27 water molecules per GC nucleotide pair and 44 per AT pair. Moreover, we noted that the fraction of nucleotides in the B form as a function of the average number of water molecules associated per base pair does not depend on the temperature. The A helical form is obtained with about 11 water molecules per nucleotide pair and this number is not very sensitive to the base composition of DNA.     

Global Conformational Dynamics of a Y-Family DNA Polymerase during Catalysis

Replicative DNA polymerases are stalled by damaged DNA while the newly discovered Y-family DNA polymerases are recruited to rescue these stalled replication forks, thereby enhancing cell survival. The Y-family DNA polymerases, characterized by low fidelity and processivity, are able to bypass different classes of DNA lesions. A variety of kinetic and structural studies have established a minimal reaction pathway common to all DNA polymerases, although the conformational intermediates are not well defined. Furthermore, the identification of the rate-limiting step of nucleotide incorporation catalyzed by any DNA polymerase has been a matter of long debate. By monitoring time-dependent fluorescence resonance energy transfer (FRET) signal changes at multiple sites in each domain and DNA during catalysis, we present here a real-time picture of the global conformational transitions of a model Y-family enzyme: DNA polymerase IV (Dpo4) from Sulfolobus solfataricus. Our results provide evidence for a hypothetical DNA translocation event followed by a rapid protein conformational change prior to catalysis and a subsequent slow, post-chemistry protein conformational change. Surprisingly, the DNA translocation step was induced by the binding of a correct nucleotide. Moreover, we have determined the directions, rates, and activation energy barriers of the protein conformational transitions, which indicated that the four domains of Dpo4 moved in a synchronized manner. These results showed conclusively that a pre-chemistry conformational change associated with domain movements was too fast to be the rate-limiting step. Rather, the rearrangement of active site residues limited the rate of correct nucleotide incorporation. Collectively, the conformational dynamics of Dpo4 offer insights into how the inter-domain movements are related to enzymatic function and their concerted interactions with other proteins at the replication fork.     

Thermophoretic melting curves quantify the conformation and stability of RNA and DNA

Measuring parameters such as stability and conformation of biomolecules, especially of nucleic acids, is important in the field of biology, medical diagnostics and biotechnology. We present a thermophoretic method to analyse the conformation and thermal stability of nucleic acids. It relies on the directed movement of molecules in a temperature gradient that depends on surface characteristics of the molecule, such as size, charge and hydrophobicity. By measuring thermophoresis of nucleic acids over temperature, we find clear melting transitions and resolve intermediate conformational states. These intermediate states are indicated by an additional peak in the thermophoretic signal preceding most melting transitions. We analysed single nucleotide polymorphisms, DNA modifications, conformational states of DNA hairpins and microRNA duplexes. The method is validated successfully against calculated melting temperatures and UV absorbance measurements. Interestingly, the methylation of DNA is detected by the thermophoretic amplitude even if it does not affect the melting temperature. In the described setup, thermophoresis is measured all-optical in a simple setup using a reproducible capillary format with only 250 nl probe consumption. The thermophoretic analysis of nucleic acids shows the technique's versatility for the investigation of nucleic acids relevant in cellular processes like RNA interference or gene silencing.     

Conformational transitions of DNA induced by changing water content of the sample: a theoretical study

A semi-phenomenological model with spatially distributed parameters is suggested to describe the processes of conformational transitions induced with change of water content of wet DNA samples. It allows describing conformational dynamics of DNA molecules with heterogeneous primary structures. It has been shown that the process of cooperative conformational transition can be simulated as propagating front of a new conformation. The evolution of a conformational perturbation of DNA molecule has been described. It can collapse for finite time or occupy the whole molecule depending on the water content of the sample.     

Snapshots of a Y-family DNA polymerase in replication: substrate-induced conformational transitions and implications for fidelity of Dpo4

Y-family DNA polymerases catalyze translesion DNA synthesis over damaged DNA. Each Y-family polymerase has a polymerase core consisting of a palm, finger and thumb domain in addition to a fourth domain known as a little finger domain. It is unclear how each domain moves during nucleotide incorporation and what type of conformational changes corresponds to the rate-limiting step previously reported in kinetic studies. Here, we present three crystal structures of the prototype Y-family polymerase: apo-Dpo4 at 1.9 Å resolution, Dpo4-DNA binary complex and Dpo4-DNA-dTMP ternary complex at 2.2 Å resolution. Dpo4 undergoes dramatic conformational changes from the apo to the binary structures with a 131° rotation of the little finger domain relative to the polymerase core upon DNA binding. This DNA-induced conformational change is verified in solution by our tryptophan fluorescence studies. In contrast, the polymerase core retains the same conformation in all three conformationally distinct states. Particularly, the finger domain which is responsible for checking base pairing between the template base and an incoming nucleotide retains a rigid conformation. The inflexibility of the polymerase core likely contributes to the low fidelity of Dpo4, in addition to its loose and solvent-accessible active site. Interestingly, while the binary and ternary complexes of Dpo4 retain an identical global conformation, the aromatic side chains of two conserved tyrosines at the nucleotide-binding site change orientations between the binary and ternary structures. Such local conformational changes may correspond to the rate-limiting step in the mechanism of nucleotide incorporation. Together, the global and local conformational transitions observed in our study provide a structural basis for the distinct kinetic steps of a catalytic cycle of DNA polymerization performed by a Y-family polymerase.     

DNA models for A, B, C and D conformations related to fiber X-ray, infrared and NMR measurements

A conformational analysis of the A, B, C and D DNA forms was made in order to establish molecular models presenting a good agreement with experimental data obtained from fiber X-ray, infrared linear dichroism and 31P NMR. The proposed models have been refined and do present good stereochemistry and optimized H-bond distances between bases associated with the Watson-Crick pairing. The DNA conformations proposed are a left handed double helix for the C form and right handed helices for A, B and D. Relations to conformational transitions between these forms are discussed.     

Real‐time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET

The numerous functions of the important class of molecular chaperones, heat shock proteins 70 (Hsp70), rely on cycles of intricate conformational changes driven by ATP‐hydrolysis and regulated by cochaperones and substrates. Here, we used Förster resonance energy transfer to study the conformational dynamics of individual molecules of Ssc1, a mitochondrial Hsp70, in real time. The intrinsic dynamics of the substrate‐binding domain of Ssc1 was observed to be uncoupled from the dynamic interactions between substrate‐ and nucleotide‐binding domains. Analysis of the fluctuations in the interdomain separation revealed frequent transitions to a nucleotide‐free state. The nucleotide‐exchange factor Mge1 did not induce ADP release, as expected, but rather facilitated binding of ATP. These results indicate that the conformational cycle of Ssc1 is more elaborate than previously thought and provide insight into how the Hsp70s can perform a wide variety of functions.     

Molecular dynamics simulations of the 136 unique tetranucleotide sequences of DNA oligonucleotides. II: sequence context effects on the dynamical structures of the 10 unique dinucleotide steps

Molecular dynamics (MD) simulations including water and counterions on B-DNA oligomers containing all 136 unique tetranucleotide basepair steps are reported. The objective is to obtain the calculated dynamical structure for at least two copies of each case, use the results to examine issues with regard to convergence and dynamical stability of MD on DNA, and determine the significance of sequence context effects on all unique dinucleotide steps. This information is essential to understand sequence effects on DNA structure and has implications on diverse problems in the structural biology of DNA. Calculations were carried out on the 136 cases embedded in 39 DNA oligomers with repeating tetranucleotide sequences, capped on both ends by GC pairs and each having a total length of 15 nucleotide pairs. All simulations were carried out using a well-defined state-of-the-art MD protocol, the AMBER suite of programs, and the parm94 force field. In a previous article (Beveridge et al. 2004. Biophysical Journal. 87:3799-3813), the research design, details of the simulation protocol, and informatics issues were described. Preliminary results from 15 ns MD trajectories were presented for the d(CpG) step in all 10 unique sequence contexts. The results indicated the sequence context effects to be small for this step, but revealed that MD on DNA at this length of trajectory is subject to surprisingly persistent cooperative transitions of the sugar-phosphate backbone torsion angles alpha and gamma. In this article, we report detailed analysis of the entire trajectory database and occurrence of various conformational substates and its impact on studies of context effects. The analysis reveals a possible direct correspondence between the sequence-dependent dynamical tendencies of DNA structure and the tendency to undergo transitions that "trap" them in nonstandard conformational substates. The difference in mean of the observed basepair step helicoidal parameter distribution with different flanking sequence sometimes differs by as much as one standard deviation, indicating that the extent of sequence effects could be significant. The observations reveal that the impact of a flexible dinucleotide such as CpG could extend beyond the immediate basepair neighbors. The results in general provide new insight into MD on DNA and the sequence-dependent dynamical structural characteristics of DNA.