Formação e estabilidade de Breathers no modelo de Peyrard-Bishop para o DNA

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Dynamic and thermodynamic anomalies of water at low temperatures: from bulk water to reverse micelles and DNA hydration layer

Liquid water is known to exhibit remarkable thermodynamic and dynamic anomalies, ranging from solvation properties in supercritical state to an apparent divergence of the linear response functions at a low temperature. Anomalies in various dynamic properties of water have also been observed in the hydration layer of proteins, DNA grooves and inside the nanocavity, such as reverse micelles and nanotubes. Here we report studies on the molecular origin of these anomalies in supercooled water, in the grooves of DNA double helix and reverse micelles. The anomalies have been discussed in terms of growing correlation length and intermittent population fluctuation of 4- and 5-coordinated species. We establish correlation between thermodynamic response functions and mean squared species number fluctuation. Lifetime analysis of 4- and 5-coordinated species reveals interesting differences between the role of the two species in supercooled and constrained water. The nature and manifestations of the apparent and much discussed liquid-liquid transition under confinement are found to be markedly different from that in the bulk. We find an interesting `faster than bulk' relaxation in reverse micelles which we attribute to frustration effects created by competition between the correlations imposed by surface interactions and that imposed by hydrogen bond network of water.

Programming chemical kinetics: engineering dynamic reaction networks with DNA strand displacement

Over the last century, the silicon revolution has enabled us to build faster, smaller and more sophisticated computers. Today, these computers control phones, cars, satellites, assembly lines, and other electromechanical devices. Just as electrical wiring controls electromechanical devices, living organisms employ "chemical wiring" to make decisions about their environment and control physical processes. Currently, the big difference between these two substrates is that while we have the abstractions, design principles, verification and fabrication techniques in place for programming with silicon, we have no comparable understanding or expertise for programming chemistry.

In this thesis we take a small step towards the goal of learning how to systematically engineer prescribed non-equilibrium dynamical behaviors in chemical systems. We use the formalism of chemical reaction networks (CRNs), combined with mass-action kinetics, as our programming language for specifying dynamical behaviors. Leveraging the tools of nucleic acid nanotechnology (introduced in Chapter 1), we employ synthetic DNA molecules as our molecular architecture and toehold-mediated DNA strand displacement as our reaction primitive.

Abstraction, modular design and systematic fabrication can work only with well-understood and quantitatively characterized tools. Therefore, we embark on a detailed study of the "device physics" of DNA strand displacement (Chapter 2). We present a unified view of strand displacement biophysics and kinetics by studying the process at multiple levels of detail, using an intuitive model of a random walk on a 1-dimensional energy landscape, a secondary structure kinetics model with single base-pair steps, and a coarse-grained molecular model that incorporates three-dimensional geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Our findings are consistent with previously measured or inferred rates for hybridization, fraying, and branch migration, and provide a biophysical explanation of strand displacement kinetics. Our work paves the way for accurate modeling of strand displacement cascades, which would facilitate the simulation and construction of more complex molecular systems.

In Chapters 3 and 4, we identify and overcome the crucial experimental challenges involved in using our general DNA-based technology for engineering dynamical behaviors in the test tube. In this process, we identify important design rules that inform our choice of molecular motifs and our algorithms for designing and verifying DNA sequences for our molecular implementation. We also develop flexible molecular strategies for "tuning" our reaction rates and stoichiometries in order to compensate for unavoidable non-idealities in the molecular implementation, such as imperfectly synthesized molecules and spurious "leak" pathways that compete with desired pathways.

We successfully implement three distinct autocatalytic reactions, which we then combine into a de novo chemical oscillator. Unlike biological networks, which use sophisticated evolved molecules (like proteins) to realize such behavior, our test tube realization is the first to demonstrate that Watson-Crick base pairing interactions alone suffice for oscillatory dynamics. Since our design pipeline is general and applicable to any CRN, our experimental demonstration of a de novo chemical oscillator could enable the systematic construction of CRNs with other dynamic behaviors.

Dynamic effects of cofactors and DNA on the oligomeric state of human mitochondrial DNA helicase

We examined the effects of cofactors and DNA on the stability, oligomeric state and conformation of the human mitochondrial DNA helicase. We demonstrate that low salt conditions result in protein aggregation that may cause dissociation of oligomeric structure. The low salt sensitivity of the mitochondrial DNA helicase is mitigated by the presence of magnesium, nucleotide, and increased temperature. Electron microscopic and glutaraldehyde cross-linking analyses provide the first evidence of a heptameric oligomer and its interconversion from a hexameric form. Limited proteolysis by trypsin shows that binding of nucleoside triphosphate produces a conformational change that is distinct from the conformation observed in the presence of nucleoside diphosphate. We find that single-stranded DNA binding occurs in the absence of cofactors and renders the mitochondrial DNA helicase more susceptible to proteolytic digestion. Our studies indicate that the human mitochondrial DNA helicase shares basic properties with the SF4 replicative helicases, but also identify common features with helicases outside the superfamily, including dynamic conformations similar to other AAA⁺ ATPases.

A Study of Ordered Gene Problems Featuring DNA Error Correction and DNA Fragment Assembly with a Variety of Heuristics, Genetic Algorithm Variations, and Dynamic Representations

Ordered gene problems are a very common classification of optimization problems. Because of their popularity countless algorithms have been developed in an attempt to find high quality solutions to the problems. It is also common to see many different types of problems reduced to ordered gene style problems as there are many popular heuristics and metaheuristics for them due to their popularity. Multiple ordered gene problems are studied, namely, the travelling salesman problem, bin packing problem, and graph colouring problem. In addition, two bioinformatics problems not traditionally seen as ordered gene problems are studied: DNA error correction and DNA fragment assembly. These problems are studied with multiple variations and combinations of heuristics and metaheuristics with two distinct types or representations. The majority of the algorithms are built around the Recentering- Restarting Genetic Algorithm. The algorithm variations were successful on all problems studied, and particularly for the two bioinformatics problems. For DNA Error Correction multiple cases were found with 100% of the codes being corrected. The algorithm variations were also able to beat all other state-of-the-art DNA Fragment Assemblers on 13 out of 16 benchmark problem instances.

Electronic couplings and on-site energies for hole transfer in DNA: systematic quantum mechanical/molecular dynamic study

The electron hole transfer (HT) properties of DNA are substantially affected by thermal fluctuations of the π stack structure. Depending on the mutual position of neighboring nucleobases, electronic coupling V may change by several orders of magnitude. In the present paper, we report the results of systematic QM/molecular dynamic (MD) calculations of the electronic couplings and on-site energies for the hole transfer. Based on 15 ns MD trajectories for several DNA oligomers, we calculate the average coupling squares 〈 V2 〉 and the energies of basepair triplets X G+ Y and X A+ Y, where X, Y=G, A, T, and C. For each of the 32 systems, 15 000 conformations separated by 1 ps are considered. The three-state generalized Mulliken-Hush method is used to derive electronic couplings for HT between neighboring basepairs. The adiabatic energies and dipole moment matrix elements are computed within the INDO/S method. We compare the rms values of V with the couplings estimated for the idealized B -DNA structure and show that in several important cases the couplings calculated for the idealized B -DNA structure are considerably underestimated. The rms values for intrastrand couplings G-G, A-A, G-A, and A-G are found to be similar, ∼0.07 eV, while the interstrand couplings are quite different. The energies of hole states G+ and A+ in the stack depend on the nature of the neighboring pairs. The X G+ Y are by 0.5 eV more stable than X A+ Y. The thermal fluctuations of the DNA structure facilitate the HT process from guanine to adenine. The tabulated couplings and on-site energies can be used as reference parameters in theoretical and computational studies of HT processes in DNA

AtTRB1, a telomeric DNA-binding protein from Arabidopsis, is concentrated in the nucleolus and shows highly dynamic association with chromatin

AtTRB1, 2 and 3 are members of the SMH (single Myb histone) protein family, which comprises double-stranded DNA-binding proteins that are specific to higher plants. They are structurally conserved, containing a Myb domain at the N-terminus, a central H1/H5-like domain and a C-terminally located coiled-coil domain. AtTRB1, 2 and 3 interact through their Myb domain specifically with telomeric double-stranded DNA in vitro, while the central H1/H5-like domain interacts non-specifically with DNA sequences and mediates protein–protein interactions. Here we show that AtTRB1, 2 and 3 preferentially localize to the nucleus and nucleolus during interphase. Both the central H1/H5-like domain and the Myb domain from AtTRB1 can direct a GFP fusion protein to the nucleus and nucleolus. AtTRB1–GFP localization is cell cycle-regulated, as the level of nuclear-associated GFP diminishes during mitotic entry and GFP progressively re-associates with chromatin during anaphase/telophase. Using fluorescence recovery after photobleaching and fluorescence loss in photobleaching, we determined the dynamics of AtTRB1 interactions in vivo. The results reveal that AtTRB1 interaction with chromatin is regulated at two levels at least, one of which is coupled with cell-cycle progression, with the other involving rapid exchange.

Dynamic Solid Phase DNA Extraction and PCR Amplification in Polyester-Toner Based Microchip

A variety of substrates have been used for fabrication of microchips for DNA extraction, PCR amplification, and DNA fragment separation, including the more conventional glass and silicon as well as alternative polymer-based materials. Polyester represents one such polymer, and the laser-printing of toner onto polyester films has been shown to be effective for generating polyester-toner (PeT) microfluidic devices with channel depths on the order of tens of micrometers. Here, we describe a novel and simple process that allows for the production of multilayer, high aspect-ratio PeT microdevices with substantially larger channel depths. This innovative process utilizes a CO(2) laser to create the microchannel in polyester sheets containing a uniform layer of printed toner, and multilayer devices can easily be constructed by sandwiching the channel layer between uncoated cover sheets of polyester containing precut access holes. The process allows the fabrication of deep channels, with similar to 270 mu m, and we demonstrate the effectiveness of multilayer PeT microchips for dynamic solid phase extraction (dSPE) and PCR amplification. With the former, we found that (i) more than 65% of DNA from 0.6 mu L of blood was recovered, (ii) the resultant DNA was concentrated to greater than 3 ng/mu L., (which was better than other chip-based extraction methods), and (iii) the DNA recovered was compatible with downstream microchip-based PCR amplification. Illustrative of the compatibility of PeT microchips with the PCR process, the successful amplification of a 520 bp fragment of lambda-phage DNA in a conventional thermocycler is shown. The ability to handle the diverse chemistries associated with DNA purification and extraction is a testimony to the potential utility of PeT microchips beyond separations and presents a promising new disposable platform for genetic analysis that is low cost and easy to fabricate.

Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer

The coordinated assembly of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polymerase holoenzyme is a multistep process. A partially opened toroid-shaped gp45 is loaded around DNA by gp44/62 in an ATP-dependent manner. Gp43 binds to this complex to generate the holoenzyme in which gp45 acts to topologically link gp43 to DNA, effectively increasing the processivity of DNA replication. Stopped-flow fluorescence resonance energy transfer was used to investigate the opening and closing of the gp45 ring during holoenzyme assembly. By using two site-specific mutants of gp45 along with a previously characterized gp45 mutant, we tracked changes in distances across the gp45 subunit interface through seven conformational changes associated with holoenzyme assembly. Initially, gp45 is partially open within the plane of the ring at one of the three subunit interfaces. On addition of gp44/62 and ATP, this interface of gp45 opens further in-plane through the hydrolysis of ATP. Addition of DNA and hydrolysis of ATP close gp45 in an out-of-plane conformation. The final holoenzyme is formed by the addition of gp43, which causes gp45 to close further in plane, leaving the subunit interface open slightly. This open interface of gp45 in the final holoenzyme state is proposed to interact with the C-terminal tail of gp43, providing a point of contact between gp45 and gp43. This study further defines the dynamic process of bacteriophage T4 polymerase holoenzyme assembly.

Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: A possible advantage of DNA over RNA as genomic material

Heteroduplex joints are general intermediates of homologous genetic recombination in DNA genomes. A heteroduplex joint is formed between a single-stranded region (or tail), derived from a cleaved parental double-stranded DNA, and homologous regions in another parental double-stranded DNA, in a reaction mediated by the RecA/Rad51-family of proteins. In this reaction, a RecA/Rad51-family protein first forms a filamentous complex with the single-stranded DNA, and then interacts with the double-stranded DNA in a search for homology. Studies of the three-dimensional structures of single-stranded DNA bound either to Escherichia coli RecA or Saccharomyces cerevisiae Rad51 have revealed a novel extended DNA structure. This structure contains a hydrophobic interaction between the 2′ methylene moiety of each deoxyribose and the aromatic ring of the following base, which allows bases to rotate horizontally through the interconversion of sugar puckers. This base rotation explains the mechanism of the homology search and base-pair switch between double-stranded and single-stranded DNA during the formation of heteroduplex joints. The pivotal role of the 2′ methylene-base interaction in the heteroduplex joint formation is supported by comparing the recombination of RNA genomes with that of DNA genomes. Some simple organisms with DNA genomes induce homologous recombination when they encounter conditions that are unfavorable for their survival. The extended DNA structure confers a dynamic property on the otherwise chemically and genetically stable double-stranded DNA, enabling gene segment rearrangements without disturbing the coding frame (i.e., protein-segment shuffling). These properties may give an extensive evolutionary advantage to DNA.

Spatiotemporal investigations of DNA damage repair using microbeams

Cellular response to radiation damage is made by a complex network of pathways and feedback loops whose spatiotemporal organisation is still unclear despite its decisive role in determining the fate of the damaged cell. Revealing the dynamic sequence of the repair proteins is therefore critical in understanding how the DNA repair mechanisms work. There are also still open questions regarding the possible movement of damaged chromatin domains and its role as trigger for lesion recognition and signalling in the DNA repair context. The single-cell approach and the high spatial resolution offered by microbeams provide the perfect tool to study and quantify the dynamic processes associated with the induction and repair of DNA damage. We have followed the development of radiation-induced foci for three DNA damage markers (i.e. γ-H2AX, 53BP1 and hSSB1) using normal fibroblasts (AG01522), human breast adenocarcinoma cells (MCF7) and human fibrosarcoma cells (HT1080) stably transfected with yellow fluorescent protein fusion proteins following irradiation with the QUB X-ray microbeam (carbon X-rays <2 µm spot). The size and intensity of the foci has been analysed as a function of dose and time post-irradiation to investigate the dynamics of the above-mentioned DNA repair processes and monitor the remodelling of chromatin structure that the cell undergoes to deal with DNA damage.

Chromatin modifications involved in the DNA damage response to double strand breaks

In eukaryotes, genomic DNA is tightly compacted into a protein-DNA complex known as chromatin. This dense structure presents a barrier to DNA-dependent processes including transcription, replication and DNA repair. The repressive structure of chromatin is overcome by ATP-dependent chromatin remodelling complexes and chromatin-modifying enzymes. There is now ample evidence that DNA double-strand breaks (DSBs) elicit various histone modifications (such as acetylation, deacetylation, and phosphorylation) that function combinatorially to control the dynamic structure of the chromatin microenvironment. The role of these mechanisms during transcription and replication has been well studied, while the research into their impact on regulation of DNA damage response is rapidly gaining momentum. How chromatin structure is remodeled in response to DNA damage and how such alterations influence DSB repair are currently significant questions. This review will summarise the major chromatin modifications and chromatin remodelling complexes implicated in the DNA damage response to DSBs.

Computational Methods for Locating and Analyzing Conserved Gene Regulatory DNA Elements

This thesis presents methods for locating and analyzing cis-regulatory DNA elements involved with the regulation of gene expression in multicellular organisms. The regulation of gene expression is carried out by the combined effort of several transcription factor proteins collectively binding the DNA on the cis-regulatory elements. Only sparse knowledge of the 'genetic code' of these elements exists today. An automatic tool for discovery of putative cis-regulatory elements could help their experimental analysis, which would result in a more detailed view of the cis-regulatory element structure and function. We have developed a computational model for the evolutionary conservation of cis-regulatory elements. The elements are modeled as evolutionarily conserved clusters of sequence-specific transcription factor binding sites. We give an efficient dynamic programming algorithm that locates the putative cis-regulatory elements and scores them according to the conservation model. A notable proportion of the high-scoring DNA sequences show transcriptional enhancer activity in transgenic mouse embryos. The conservation model includes four parameters whose optimal values are estimated with simulated annealing. With good parameter values the model discriminates well between the DNA sequences with evolutionarily conserved cis-regulatory elements and the DNA sequences that have evolved neutrally. In further inquiry, the set of highest scoring putative cis-regulatory elements were found to be sensitive to small variations in the parameter values. The statistical significance of the putative cis-regulatory elements is estimated with the Two Component Extreme Value Distribution. The p-values grade the conservation of the cis-regulatory elements above the neutral expectation. The parameter values for the distribution are estimated by simulating the neutral DNA evolution. The conservation of the transcription factor binding sites can be used in the upstream analysis of regulatory interactions. This approach may provide mechanistic insight to the transcription level data from, e.g., microarray experiments. Here we give a method to predict shared transcriptional regulators for a set of co-expressed genes. The EEL (Enhancer Element Locator) software implements the method for locating putative cis-regulatory elements. The software facilitates both interactive use and distributed batch processing. We have used it to analyze the non-coding regions around all human genes with respect to the orthologous regions in various other species including mouse. The data from these genome-wide analyzes is stored in a relational database which is used in the publicly available web services for upstream analysis and visualization of the putative cis-regulatory elements in the human genome.

Acetylation of Transition Protein 2 (TP2) by KAT3B (p300) Alters Its DNA Condensation Property and Interaction with Putative Histone Chaperone NPM3

The hallmark of mammalian spermiogenesis is the dramatic chromatin remodeling process wherein the nucleosomal histones are replaced by the transition proteins TP1, TP2, and TP4. Subsequently these transition proteins are replaced by the protamines P1 and P2. Hyperacetylation of histone H4 is linked to their replacement by transition proteins. Here we report that TP2 is acetylated in vivo as detected by anti-acetylated lysine antibody and mass spectrometric analysis. Further, recombinant TP2 is acetylated in vitro by acetyltransferase KAT3B (p300) more efficiently than by KAT2B (PCAF). In vivo p300 was demonstrated to acetylate TP2. p300 acetylates TP2 in its C-terminal domain, which is highly basic in nature and possesses chromatin-condensing properties. Mass spectrometric analysis showed that p300 acetylates four lysine residues in the C-terminal domain of TP2. Acetylation of TP2 by p300 leads to significant reduction in its DNA condensation property as studied by circular dichroism and atomic force microscopy analysis. TP2 also interacts with a putative histone chaperone, NPM3, wherein expression is elevated in haploid spermatids.Interestingly, acetylation of TP2 impedes its interaction with NPM3. Thus, acetylation of TP2 adds a new dimension to its role in the dynamic reorganization of chromatin during mammalian spermiogenesis.