Sequence- and structure-dependent DNA base dynamics: synthesis, structure, and dynamics of site and sequence specifically spin-labeled DNA

A nitroxide spin-labeled analogue of thymidine (1a), in which the methyl group is replaced by an acetylene-tethered nitroxide, was evaluated as a probe for structural and dynamics studies of sequence specifically spin-labeled DNA. Residue 1a was incorporated into synthetic deoxyoligonucleotides by using automated phosphite triester methods. 1H NMR, CD, and thermal denaturation studies indicate that 1a (T*) does not significantly alter the structure of 5'-d(CGCGAATT*CGCG) from that of the native dodecamer. EPR studies on monomer, single-stranded, and duplexed DNA show that 1a readily distinguishes environments of different rigidity. Comparison of the general line-shape features of the observed EPR spectra of several small duplexes (12-mer, 24-mer) with simulated EPR spectra assuming isotropic motion suggests that probe 1a monitors global tumbling of small duplexes. Increasing the length of the DNA oligomers results in significant deviation from isotropic motion, with line-shape features similar to those of calculated spectra of objects with isotropic rotational correlation times of 20-100 ns. EPR spectra of a spin-labeled GT mismatch and a T bulge in long DNAs are distinct from those of spin-labeled Watson-Crick paired DNAs, further demonstrating the value of EPR as a tool in the evaluation of local dynamic and structural features in macromolecules.     

Heterogeneity of double-stranded DNA local structure and dynamics: ultrasound studies

A method for studying the local inhomogeneities of DNA and its dynamics is proposed. The method is based on the combination of two procedures, splitting the DNA molecules by ultrasound and analysis of DNA fragments obtained by gel electrophoresis. The frequency of cleavage of internucleotide bonds was found to depend on the type of nucleotides forming the bond and on their nearest neighbors. Estimates of cleavage frequencies in each of 16 dinucleotides showed that, in the 5'-d(CpG)-3', 5'-d(CpA)-3', and 5'-d(CpT)-3' dimers, the cleavage occurs considerably more frequently than in the rest, and the frequency of cleavage depends on the nearest neighbors. It was shown that the double-helix cleavage can occur with shifts by several nucleotides. Physical prerequisites were considered that can lead to this pattern of sequence - specific cleavage.     

AFM for analysis of structure and dynamics of DNA and protein-DNA complexes

This paper describes protocols for studies of structure and dynamics of DNA and protein-DNA complexes with atomic force microscopy (AFM) utilizing the surface chemistry approach. The necessary specifics for the preparation of functionalized surfaces and AFM probes with the use of silanes and silatranes, including the protocols for synthesis of silatranes are provided. The methodology of studies of local and global conformations DNA with the major focus on the time-lapse imaging of DNA in aqueous solutions is illustrated by the study of dynamics of Holliday junctions including branch migration. The analysis of nucleosome dynamics is selected as an example to illustrate the application of the time-lapse AFM to studies of dynamics of protein-DNA complexes. The force spectroscopy is the modality of AFM with a great importance to various fields of biomedical studies. The AFM force spectroscopy approach for studies of specific protein-DNA complexes is illustrated by the data on analysis of dynamics of synaptic SfiI-DNA complexes. When necessary, additional specifics are added to the corresponding example.     

Solution structure and dynamics of GCN4 cognate DNA: NMR investigations

A 12 bp long GCN4-binding, self-complementary duplex DNA d(CATGACGTCATG)₂ has been investigated by NMR spectroscopy to study the structure and dynamics of the molecule in aqueous solution. The NMR structure of the DNA obtained using simulated annealing and iterative relaxation matrix calculations compares quite closely with the X-ray structure of ATF/CREB DNA in complex with GCN4 protein (DNA-binding domain). The DNA is also seen to be curved in the free state and this has a significant bearing on recognition by the protein. The dynamic characteristics of the molecule have been studied by ¹³C relaxation measurements at natural abundance. A correlation has been observed between sequence-dependent dynamics and recognition by GCN4 protein.     

Nucleosome structure relaxation during DNA unwrapping: Molecular dynamics simulation study

Effects of local relaxation of the nucleosome structure after DNA unwrapping from the histone octamer are considered in this paper. The influence of charge distribution in histones on the kinetics of DNA rewrapping was studied. It was shown that ionic environment rapidly stabilizes during relaxation simulation of the system by molecular dynamics. In the case of short relaxation, a rapid irreversible restoration of the structure, which is similar to a crystal one, occurs. In the case of longer relaxation, DNA rewrapping does not occur despite the absence of apparent differences in the ionic environment of DNA. The change in the quadrupole moment of the system during relaxation was shown.     

Visualizing the dynamics of chromosome structure formation coupled with DNA replication

A basic question of cell biology is how DNA folds to chromosome. Numbers of examples have suggested the involvement of DNA replication in chromosome structure formation. To visualize and identify the dynamics of chromosome structure formation and to elucidate the involvement of DNA replication in chromosome construction, Cy3-2′-deoxyuridine-5′-triphosphate direct-labeled active replicating DNA was observed in prematurely condensed chromosomes (PCCs) under a confocal scanning microscope utilized with drug-induced premature chromosome condensation (PCC) technique that facilitates the visualization of interphase chromatin as condensed chromosome form. S-phase PCCs revealed clearly the drastic dynamics of chromosome formation that transits during S-phase from a ‘cloudy nebula’ to numerous numbers of ‘beads on a string’ and finally to ‘striped arrays of banding structured chromosome’ along with the progress of DNA replication. The number, distribution, and shape of replication foci were also measured in individual subphases of S-phase more precisely than reported previously; maximally, ∼1,400 foci of 0.35 μm average radius size were scored at the beginning of the S-phase, and the number reduced to ∼100 at the end of the S-phase. Drug-induced PCC clearly provided the new insight that eukaryote DNA replication is tightly coupled with the chromosome condensation/compaction for the construction of the higher-ordered structure of the eukaryote chromosome.     

A note on graphing helical parameters of dynamics structure of DNA

A graphical procedure for analysis of helical parameters in dynamic structure of DNA is described. The performance of the procedure is illustrated by analysis of a 20 ps dynamics simulation of the non-self-complementary ninemer, 5'CAAACAGGA:5'TCCTGTTTG, which is a part of DNA from lacI gene. The dynamics trajectory of the duplex shows sequence-dependent fluctuations of helical parameters.     

DNA conformation and dynamics

Nucleotide conformation and dynamics are important for the study of radiation damage to DNA at the atomic level. It is necessary to study not only normal oligonucleotide structure but also those containing modified bases which result from interaction with OH-radicals. There are now over 8000 atomic coordinate entries in the Brookhaven Protein Data Bank, of which over 900 relate to experimentally determined structures of nucleic acids and nucleic acid/protein complexes. We review some of these data which have led to the elucidation of novel DNA conformations, insight into DNA sequence specificity and knowledge of protein/DNA interactions. Further understanding of the conformation, stability and dynamics of nucleic acids has come from molecular modelling. We have used such techniques to study chemical modifications to bases such as alkylation of thymine and guanine and the effects of curvature in longer sequences. Recent improvements in this area include the inclusions of explicit counter-ions and solvent molecules, the use of Particle Mesh Ewald methods to incorporate the long-range electrostatic interactions and the use of longer time scale simulations. We have employed these methods to analyse the effects of incorporation of 8-oxodeoxyguanosine into duplex DNA. This lesion is a common result of radiation damage and is known to have important effects in mutagenesis, cancer and ageing. Received: 7 October 1998 / Accepted in revised form: 18 January 1999     

Distinctive features in the structure and dynamics of the DNA repeat sequence GGCGGG

G-rich DNA has been known to form a variety of folded and multistranded structures, with even single base modifications causing important structural changes. But, very little is known about the dynamic characteristics of the structures, which may play crucial roles in facilitating the structural transitions. In this background, we report here NMR investigations on the structure and dynamics of a DNA repeat sequence GGCGGG in aqueous solution containing Na+ ions at neutral pH. The chosen sequence d-TGGCGGGT forms a parallel quadruplex with a C-tetrad in the middle, formed by symmetrical pairing of four Cs in a plane via NH2-O2 H-bonds. 13C relaxation measurements at natural abundance for C' sugar carbons provided valuable insight into the sequence specific dynamism of G and C-tetrads in the quadruplex. The C4 tetrad seems to introduce high conformational dynamism at milli- to micro-second time scale in the quadruplex. Concomitantly, there is a decrease in the pico-second time scale dynamics. Interestingly, these effects are seen more prominently at the G-tetrads on the 3' end of C-tetrad than on its 5' end. These observations would have important implications for the roles the tetrads may play in many biological functions.     

Apolipoprotein structure and dynamics

PURPOSE OF REVIEW: This review highlights recent advances in structural studies of exchangeable human apolipoproteins and the insights they provide into lipoprotein action in cardiovascular and amyloid diseases. RECENT FINDINGS: The high-resolution X-ray crystal structure of free apoA-II reveals a parallel helical array that may represent other lipid-poor apolipoproteins, and the structure in complex with detergent substantiates the belt model for the protein arrangement on lipoproteins. Nuclear magnetic resonance structures of apolipoprotein-detergent complexes show a repertoire of curved helical conformations, suggesting multiple helical arrangements on the lipid. Low-resolution spectroscopic analyses, interface studies and molecular modeling provide new insights into the 'hinge-domain' mechanism of apolipoprotein adaptation at variable lipoprotein surfaces. A kinetic mechanism for lipoprotein stabilization is proposed. SUMMARY: Cumulative evidence supports the belt model that provides a general structural basis for understanding the molecular mechanisms of functional apolipoprotein reactions, such as binding to lipoprotein receptors, lipid transporters, and the activation of lipophilic enzymes. However, the detailed protein and lipid conformations on lipoproteins and the underlying molecular interactions are unclear. New insights will hopefully emerge once the first detailed lipoprotein structure is solved.