Feb 14, 2017 - method to probe the coordination between changes in DNA conformation and reconfiguration of the histone core. We used time-resolved small ...
Tuesday, February 14, 2017 method to probe the coordination between changes in DNA conformation and reconfiguration of the histone core. We used time-resolved small angle x-ray scattering (TR-SAXS) to visualize DNA unwrapping and time-resolved Fo¨rster resonance energy transfer (TR-FRET) to monitor dimer release during the saltinduced disassembly of nucleosomes. Applying this approach to study the formation of the hexasome and tetrasome (missing one or two H2A-H2B dimers, respectively), we found that the asymmetric unwrapping of the DNA guides the sequential release of proteins. This link between DNA conformation and histone composition may provide new mechanistic insight into the activity of chromatin remodelers and histones chaperones. 1826-Pos Board B146 Sequence Dependence in Salt Based Nucleosome Unwrapping Using Saxs Alexander Mauney1, Joshua Tokuda1, Yujie Chen1, Lisa Gloss2, Traci Topping2, Oscar Gonzalez3, Lois Pollack1. 1 Cornell University, Ithaca, NY, USA, 2Washington State University, Pullman, WA, USA, 3University of Texas at Austin, Ithaca, NY, USA. Wrapping and unwrapping of DNA from nucleosome core particles is a major part of DNA dynamics in vivo, though there is much about it that is still unknown. As DNA availability is a key component of transcription regulation, understanding how variations in DNA sequence and histone structure or composition impact their binding is crucial for understanding the overall transcription process, and how DNA is organized into chromatin. In this work, small angle x-ray scattering (SAXS) was used to examine multiple combinations of DNA sequence and histone variants at different salt concentrations, and in solution where there are no geometric constraints. By using salt to destabilize the nucleosome-DNA complex multiple unwrapped states are accessible, allowing for many possible pathways to be explored by the complexes. We use novel Monte-Carlo methods to generate realistic sequence-dependent unwrapped structures for the nucleosomal DNA. These structures are used in ensemble optimization methods to determine which ones are represented in the SAXS data1. We determine differences in both the unwrapping paths and equilibrium constants characteristic of the different histone/DNA combinations. The changes in the pathway are related to structural differences between the DNA sequences. 1. Chen Y., Tokuda J.M., Topping T., Sutton J.L., Meisburger S.P., Pabit S.A., Gloss L.M., Pollack L. Revealing transient structures of nucleosomes as DNA unwinds. Nucleic Acids Res. 2014;42:8767-8776. 1827-Pos Board B147 Investigating the Histone Replacement Pathway in Sperm Using TPM Elizabeth D. White, Adam Smith, Obinna Ukogu, Hilary Bediako, Moumita Dasgupta, Ashley Carter. Physics, Amherst College, Amherst, MA, USA. DNA is folded in drastically different ways in somatic cells and sperm cells. In somatic cells, DNA is wrapped around histone proteins and remains accessible for transcription and replication. However, in sperm cells, DNA is dramatically condensed into a toroid structure when the histones are replaced by protamine proteins. Here, we investigate the process of histone replacement by protamine using tethered particle motion (TPM). TPM measurements are ideal for looking at DNA folding since measurements are taken at zero force. In TPM, one end of the DNA is attached to a glass coverslip and the other to a 1-micron-diameter bead to form a tether. We record the motion of the bead using video microscopy and relate this motion to the length of the DNA with about 10 nm precision. The length of the DNA changes as histones are unwrapped from the DNA and replaced by protamine, allowing us to reconstruct the folding pathway for histone replacement, as well as the energy landscape. This pathway is important in research on fertility, epigenetics, and biomaterials research. 1828-Pos Board B148 The Molecular Mechanism of Nucleosome Breathing David Winogradoff, Aleksei Aksimentiev. Center for the Physics of Living Cells, Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA. Meters of DNA wrap around histone proteins to form nucleosomes and fit inside the micron-diameter nucleus. In order for DNA to become available for template-directed processes—such as transcription, replication, and repair— nucleosomes must unwrap and disassemble. Single-molecule studies indicate that the outer stretches of nucleosomal DNA spontaneously unwrap and rewrap from the histone core, a process known as ‘‘DNA breathing.’’ Using the explicit solvent molecular dynamics method, we observed, for the first time, spontaneous unwrapping of DNA at ultimate, atomic-scale resolution. Starting from a crystal structure of a nucleosome particle, we built several variants of solvated nucleosome systems that differed from one another only by the nucleotide sequence of the DNA and/or the ionic conditions. At physiological salt conditions, the DNA was observed to maintain its highly bent conformation, stabilized by the contacts between the charged amino
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acids of the histone proteins and the DNA minor groove. At an elevated salt condition, however, the ends of the nucleosomal DNA were observed to spontaneously detach from and reattach to the histone core in discrete 10.5 basepair steps. We found the breakage of histone-DNA contacts to occur in several steps: a positive histone side chain first abruptly exits the minor groove, forms a salt bridge with a DNA backbone phosphate for an extended period of time, and then the contact breaks completely. Reattachment of the DNA proceeds through the same steps in the reverse order. Finally, we find the sequence of the DNA to influence the likelihood of spontaneous disassociations, suggesting that certain segments of DNA could be more likely to initiate transcription and more accessible to chromatin binding factors. Our studies demonstrate the utility of large, microsecond timescale atomistic simulations for characterizing spontaneous processes that are beyond the resolution of single-molecule experimental methods. 1829-Pos Board B149 DNA in Tight Spaces: Linking Structure, Stability and Protection in Protamine Packaged DNA Jason E. DeRouchey. Chemistry, University of Kentucky, Lexington, KY, USA. Packaged DNA is ubiquitous in nature and the laboratory with examples ranging from chromatin, viruses, sperm cells, bacterial nucleoids, artificial viruses and gene therapy constructs. Sperm nuclei are one of the best examples of in vivo maximum DNA compaction and therefore an ideal model system to study biophysically. Despite intense research, the physical mechanisms underlying tight packaging of DNA remain poorly understood especially at the molecular level. Spermiogenesis is a unique multi-step process resulting ultimately in the replacement of histones by protamines in sperm nuclei to a final volume roughly 1/20th that of a somatic nucleus. The near crystalline organization of DNA in mature sperm is thought crucial for both DNA delivery and the protection of genetic information due to the absence of DNA repair. In this talk, I will first discuss our past studies on understanding how cations modulate DNA-DNA forces in the condensed phase and the interrelationships between cation chemistry, packaging densities and compaction. The last half of my talk will discuss recent experiments aimed at understanding the various biological implications for both protamine-DNA packaging and correlations to infertility and oxidative stress in sperm chromatin. 1830-Pos Board B150 Investigating the Mechanics of Protamine-Induced DNA Condensation in Sperm Hilary Bediako, Adam D. Smith, Obinna Ukogu, Moumita Dasgupta, Elizabeth White, Ashley R. Carter. Physics & Astronomy, Amherst College, Amherst, MA, USA. We examine how protamine, a positively-charged nuclear protein, causes DNA compaction within the nucleus of a sperm cell. Beyond the basic science of understanding the mechanics behind DNA condensation, DNA folding in sperm has implications in epigenetics, fertility, and material science. In order to study how DNA folds in sperm cells, we perform an in vitro Tethered Particle Motion (TPM) assay to look at the folding of individual DNA molecules. In TPM, a polystyrene bead is attached to a DNA molecule, which is tethered to a coverslip. By observing the position of the bead over time with video microscopy, we can measure the length of the DNA to a precision of about 10 nm. When protamine binds to the DNA, it forms approximately 60-nm-diameter loops, which then condense into toroids. Here, we see individual looping events that indicate that protamine-induced toroid formation is a stepwise mechanism. 1831-Pos Board B151 Holliday Junction Structure Development for Single-Molecule Visualization Mate Gyimesi, Zoltan Kovacs, Mihaly Kovacs. Biochemistry, Eotvos University, Budapest, Hungary. A double Holliday junction (DHJ) DNA structure is formed as a key intermediate during homologous recombination (HR), supporting error-free somatic DNA repair of double-stranded DNA breaks and the meiotic formation of gametes. In HR, the broken DNA ends are processed and homologous DNA serves as template. DHJ structure is formed when both processed ends are engaged. This structure can either be resolved by nucleolytic cleavage (prominent during meiosis) or dissolved by the concerted action of RecQ-family helicases (e.g. RecQ, Sgs1, BLM), type I topoisomerases (Top3a, TOP3A) and eukaryotic regulatory proteins (RMI1 and RMI2), called the BTR complex in humans. The BTR complex migrates the HJ branches and decatenates the final structure. This process is necessary to avoid chromosomal rearrangements in somatic cells as it solely results in non-crossover products. Although the protein components necessary for DHJ dissolution have been described, little is known about the molecular/biophysical mechanism of HJ migration. Investigation of these mechanisms requires singlemolecule techniques to reveal otherwise hidden properties (e.g. processivity,
