# Workshop Programme

## for period 3 - 7 September 2012

### Topological Aspects of DNA Function and Protein Folding

3 - 7 September 2012

Timetable

 Wednesday 5 September 08:30-09:10 Benham, C (University of California, Davis, USA) Superhelically Driven Structural Transitions in Genomic DNA - Theoretical Analyses, Genomic Distributions and Roles in Regulation Sem 1 DNA is known to be a highly polymorphic molecule, capable of assuming several alternate conformations in addition to the standard Watson-Crick B-form. These include states of strand separation, left handed helices, cruciforms, and three- and four-stranded structures. Although the B-form is its default conformation in vivo, regions within genomic DNA can be driven into alternate structures by the superhelicity imposed on the molecule by enzymatic activities and by transcription. This talk will present theoretical analyzes of several types of transitions, and of competitions among them. The predicted genomic distributions of locations susceptible to different types of superhelical transitions will be shown, and their statistical significances will be assessed. Several situations will be described where transitions to alternate structures serve biological roles in either normal or pathological processes. 09:10-09:50 Dorman, C (Trinity College Dublin, Ireland) Co-operative roles for DNA supercoiling and nucleoid-associated proteins in the regulation of bacterial transcription Sem 1 DNA supercoiling and nucleoid-associated proteins (NAPs) contribute to the regulation of transcription of many bacterial genes. The horizontally-acquired Salmonella pathogenicity island (SPI) genes respond positively to DNA relaxation, to the Fis and H-NS nucleoid-associated proteins and to the OmpR global regulatory protein. The ompR gene is autoregulated and responds positively to DNA relaxation. Binding of the Fis and OmpR proteins to DNA in the SPI-1 and SPI-2 islands and at the ompR gene promoter is differentially sensitive to the topological state of the DNA while H-NS binds regardless of the topological state of the DNA. These data illustrate the overlapping and complex nature of NAP and DNA topological contributions to transcription control in bacteria. They also show that these properties are shared by the core and the horizontally-acquired components of the bacterial genome. 09:50-10:10 Morning Coffee 10:10-10:50 Travers, A (University of Cambridge) Chromosomes as topological machines-the role of DNA thermodynamics Sem 1 The chirality of the DNA molecule underpins its ability to partition superhelicity between twist and writhe. We argue that manipulation of superhelical density and of partitioning by topological devices and processive ATP-dependent motors (DNA and RNA polymerases and topoisomerases) is a fundamental property of both bacterial and eukaryotic chromosomes. On this view chromosomes act as machines in which topological transitions operate at several functional levels - local (e.g. transcription initiation sites), regional (constrained superhelical domains) and global (chromosomes) levels. The partition between twist and writhe is dependent in part on the sequence of DNA. We have shown that in the E. coli chromosome gradients of DNA gyrase binding sites from the origin to the terminus of DNA replication along both replichores correlate with temporal patterns of gene expression during the growth cycle such that genes expressed during exponential growth are preferentially located in the Ori-proximal region. These observations imply that during exponential growth there exist gradients of superhelical density from the origin to the terminus. Intriguingly the chromosomal DNA sequences exhibit, on average, a gradient of DNA stacking energy in the same direction. We argue that this gradient in the physicochemical properties of DNA integrates the functional response to changes in superhelical density and to regulation by abundant nucleoid-associated proteins. We further show that the genetic and chromatin organisation in yeast chromatin assembled both in vitro and in vivo is highly dependent on, the stacking/melting energies of DNA sequences. The regions of chromosomes that are sites for topological manipulation (such as transcription and replication initiation sites and preferred sites for topoisomerase II) correlate strongly with low stacking energies and high flexibility. Such regions concomitantly exhibit low nucleosome occupancy. We conclude that the most flexible DNA sequences are, counter-intuitively, poor substrates for octamer deposition. In contrast high nucleosome occupancy correlates with DNA sequences of moderately high stacking energies. In such relatively stiff sequences positioned nucleosomes can often be related to a bending anisotropy appropriate for nucleosome formation. 10:50-11:30 Zechiedrich, L (Baylor College of Medicine, Houston, Texas, USA) How DNA topology and DNA length affect the body's defense against nucleic acids of invading organisms in the blood Sem 1 It has long been known that human blood contains enzymes that digest DNA to protect the body against invasion by foreign organisms. We set out to determine how DNA length and supercoiling affected DNA vector survival in human serum. Closed circular, supercoiled vectors ranging from ~300 to ~4,000 bp were incubated at 37°C in human serum. Aliquots were taken over several days and were analyzed by gel electrophoresis. We found that digestion in human serum strongly correlated with increasing DNA length. To our surprise, we also uncovered a trend by which serum proteins bound and protected DNA. We recently published that the compaction by DNA supercoiling protected small (<1,200 bp) DNA circles from the mechanical shear forces of aerosolization or sonication (Catanese et al. 2012); we hypothesized that a similar trend would be observed with human serum degradation. This hypothesis proved incorrect because linear and nicked DNA survived ~ 3- to 6-fold longer than supercoiled DNA. These results agree with previously published data showing that DNAse I, the major nuclease in human blood, preferentially acts on supercoiled DNA. Together, these data support a model in which foreign DNA is gapped by nucleases in the bloodstream and that this action is enough to hamper replication and transcription of DNA vectors in human cells. In addition to explaining how the blood protects humans from invasions, this understanding opens the door for designing DNA that is relaxed, closed circular, and tiny for gene therapy to be delivered intravenously. The lack of supercoiling allows the circles to persist longer and reach their target cells where supercoiling will then be restored, resulting in transcription and gene expression. This work was supported by NIH RO1AI054830, Human Frontier Science Program, and Seattle's Children's Hospital Research Foundation, part of NGEC, to L.Z. T.J.B. was supported by NIH Grant T32 GM88129. 11:30-11:50 Chirikjian, G S (Johns Hopkins University) Twisted paths in Euclidean groups: Keeping track of total orientation while traversing DNA Sem 1 This talk introduces a new mathematical structure for modeling global twist in DNA. The relative rigid-body motion between reference frames attached either to a backbone curve, bi-rods, or individual bases in DNA, can be described well using elements of the Euclidean motion group, SE(n). However, the group law for Euclidean motions does not keep track of overall twist. In the planar case, the universal covering group of SE(2) identifies orientation angle as a quantity on the real line rather than on the circle, and hence keeps track of global'' rotations (not modulo 360 degrees). However, in the three-dimensional case, no such structure exists since the the orientational part of the universal cover of SE(3) can be identified with the quaternion sphere. In this talk a new mathematical structure for adding'' framed curves and extracting global twist is present. Though reminiscent of the group operation in braid theory and in homotopy theory, this structure is distinctly different, as it is geometric in nature, rather than topological. The motivation for this mathematical structure and its applications to DNA conformation will be presented. 11:50-12:10 Cortini, R (Imperial College London) Chiral effects in DNA supercoiling Sem 1 Supercoiling is a topological property of DNA which is known to be crucially important in the genetic regulation of virtually every living cell. Electrostatic interactions play a fundamental role in determining the conformation of DNA molecules. They are generally taken into account assuming that the charge is homogeneously smeared on the surface of DNA molecules. We developed a theory that instead takes into account the helical pattern of charge on the DNA molecular surfaces. We find that the intrinsic chirality of the charge structures gives rise to important and non trivial phenomena. Crucially, it determines an asymmetry in the energetics of DNA-DNA crossovers: right-handed crossings, occurring in positively supercoiled molecules are more stable than left-handed ones, which occur in negatively supercoiled molecules. We explored the consequences of this fact first by developing a theory of spontaneous DNA braid formation, and then applying it to closed loop DNA supercoilin g and single-molecule DNA micromanipulation experiments. The theory can give an account of some yet unexplained observations and biological facts. It gives a plausible explanation for the occurrence of tight supercoiling of DNA loops observed in cryo-EM and AFM images in high ionic strength environment. It can shed light on the preference for positive supercoiling in hyperthermophylic bacteria and archea. Finally, it induces to reinterpret classical experiments that show that divalent metal ions overwind DNA. The biological implications of these important facts could be very important, and are yet to be fully explored. 12:30-13:30 Lunch at Wolfson Court 13:30-14:10 Maddocks, J (EPFL, Lausanne, Switzerland) Sequence-Dependent Coarse-Grain Descriptions of DNA: models, methods and simulations Sem 1 14:10-14:50 Zakrzewska, K (BPC, Paris, France) DNA recognition studied by molecular simulations Sem 1 In order to fulfill its biological role DNA has to interact with other molecules, ranging from small ligands to protein complexes. In many cases these molecules intercalate, at least partially, into DNA. This intercalation can involve the conjugated rings of a drug, or the hydrophobic side chains of a protein. How different DNA binding molecules find their target sites, and what role intercalation plays in this mechanism, is still not understood. We try to answer these questions by analyzing the energetic and mechanistic aspects of recognition using molecular simulations at the atomic level. Results will be presented for binding a small drug, daunomycin, and for binding a protein, SRY, a mammalian transcription factor. We will show that daunomycin intercalates into DNA by a complex, multistep process, starting with an intermediate, minor groove bound state. In the case of SRY, the mechanism of DNA sequence specificity, via deformation of the double helix, will be discussed. References: A systematic molecular dynamics study of nearest-neighbor effects on base pair and base pair step conformations and fluctuations in B-DNA, R.Lavery et al. Nucl. Acids Res. (2010) 38(1): 299-313 Protein–DNA Recognition Triggered by a DNA Conformational Switch, B. Bouvier et al, Angew. Chem. Int. Ed. 2011, 50, 6516 –6518 Multistep Drug Intercalation: Molecular Dynamics and Free Energy Studies of the Binding of Daunomycin to DNA, M. Wilhelm et al, JACS (2012), published on line. 14:50-15:20 Afternoon Tea and Poster Session 15:20-15:40 Baxter, J (University of Sussex) The yeast Pif1 family helicase RRM3 promotes DNA unwinding during replisome swivelling Sem 1 During termination of DNA replication, replisomes overcome the topological tension that occurs as forks converge by coupling final unwinding with fork swivelling. Here I show that cells lacking the DNA helicase RRM3 accumulate terminating late replication intermediates (LRI) in plasmids both with and without characterised pause sites. Rrm3 deletion does not alter the level of swivelling that occurs during termination but its depletion extends the lifetime of LRI while it’s over-expression leads to the rapid unwinding of the LRI. Therefore RRM3 promotes DNA unwinding when the replisome swivels during termination. Potentially, this activity is also generally utilised during DNA replication to bypass topological blocks. 15:40-16:00 Hanke, A (University of Texas at Brownsville, USA) Denaturation transition of stretched DNA in the presence of DNA-binding ligands Sem 1 Stretching experiments on DNA in the presence of DNA binding ligands have been shown to reveal insight into biological processes of DNA-ligand binding. We generalize the Poland-Scheraga model to consider DNA denaturation in the presence of an external stretching force and DNA-binding ligands which bind to double-stranded DNA by intercalating between two adjacent base pairs. We obtain the phase diagram of DNA denaturation as a function of temperature, stretching force, and the chemical potential of the DNA-binding ligand. Force-extension relations are compared with recent DNA stretching experiments in the presence of DNA intercalating ethidium and ruthenium complexes. 16:00-16:20 Bohr, J (DTU, Denmark) Twist neutrality and topological aspects of nucleosomal DNA Sem 1 16:20-17:00 Swigon, D (University of Pittsburgh, USA) Dynamics of DNA supercoiling and knotting Sem 1 Recent experiments on electrostatically induced migration of DNA in nanochanels reveal an intricate phenomenon of compaction of migrating DNA that promotes knotting of the molecule. Subsequent relaxation of the molecule proceeds along several distinct kinetic regimes. The structural details of DNA configurations in different stages of the process are yet unknown. We investigate this and other related phenomena of DNA dynamics using a model in which DNA is represented by a charged elastic rod immersed in a viscous incompressible fluid and the governing equations of the system are solved numerically using the generalized immersed boundary method. The equations of motion of the rod include the fluid–structure interaction, rod elasticity and a combination of two interactions that prevent self-contact, namely the electrostatic interaction and hard-core repulsion. Presented will be results on the effects of electrostatics, steric repulsion, and thermal fluctuations on DNA supe rcoiling and knotting dynamics. 17:00-17:40 Olson, W (Rutgers University, New Jersey, USA) Simulated looping propensities of protein-decorated DNA Sem 1 Although the genetic messages in DNA are stored in a linear sequence of base pairs, the genomes of living species do not function in a linear fashion. Gene expression is regulated by DNA elements that often lie far apart along the genomic sequence but come close together during genetic processing. The intervening residues form loops, which are organized by the binding of various proteins. For example, in E. coli the Lac repressor protein assembly binds two DNA operators, separated by 92 or 401 base pairs, and suppresses the formation of gene products involved in the metabolism of lactose. The system also includes several highly abundant architectural proteins, such as Fis and HU, which, upon binding, bend a double-helical turn of DNA by 45 degrees or more. In order to gain a better understanding of the mechanics of DNA looping, we have investigated the effects of various proteins on the configurational properties of fragments of DNA, treating the DNA with elastic potentials t hat consider the intrinsic structure and deformability of successive base pairs and incorporating the known three-dimensional structural effects of various proteins on DNA double-helical structure. The presentation will highlight some of the new models and computational techniques that we have developed to generate the three-dimensional configurations of protein-mediated DNA loops and illustrate new insights gained from this work about the effects of various proteins on DNA topology and the apparent contributions of non-specific binding proteins to gene expression. 17:40-19:00 Poster Session 19:30-22:00 Conference Dinner at Christ's College