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Effects of Salt on the Stability of a G-Quadruplex from the Human c-MYC Promoter
Tuesday, March 1, 2016 then analyzed in multiple ways, including calculating first passage time distributions between WC and HG configurations, and determination of regions in configuration space of maximum flux. In order to make the system numerically tractable for a Fokker-Planck approach to diffusion, the energy surface was also fit to a simpler 2D analytical function with a 20 term Fourier series solely in the ‘‘flipover’’ dimension and a separate harmonic term approximating the ‘‘flip-out’’ dimension. Both approaches were utilized to determine favorable pathways for WC/HG transitions, and their respective time scales. 2000-Pos Board B144 Linker Histones and the Dynamic Chromatin Fiber Stefjord Todolli, Nicolas Clauvelin, Wilma K. Olson. Center for Quantitative Biology, Rutgers University, Piscataway, NJ, USA. DNA in eukaryotic cells is highly compacted into a hierarchical chromatin structure to fit inside the nucleus. Linker histones play an important role in this packing. Their interaction with the nucleosomes and intervening DNA linkers, in combination with linker length, are believed to affect chromatin folding and long-range interactions. The details of chromatin structure at this level of compaction are still an open question but have profound implications on biological processes, such as gene expression. We have constructed a complete coarse-grained chromatin model from recently ˚ resolution electron microscopy maps of two chromatin fibers published 11-A (Song et al., 2014). The arrangements of the modeled DNA linkers point to four or more distinct modes of linker histone-nucleosome association that provide hints of the local dynamic structure. We use these association modes as well as a recently published crystal structure of the chromatosome complex (Zhou et al., 2015) in coarse-grained Monte Carlo simulations of precisely positioned nucleosomal arrays. The states of chromatin captured in our simulations reveal how the presence and positioning of linker histones, in combination with varying DNA linker lengths, affect long-range chromatin communication and the geometry of the chromatin fiber. 2001-Pos Board B145 Entropy Calculations of Hoogsteen and Watson-Crick Conformations James McSally, Ioan Andricioaei. U of California, Irvine, Irvine, CA, USA. The conformations that DNA takes are often vital in many biological processes. The physical effects Hoogsteen conformation base pairs have on the full strand of DNA are still being determined. Through molecular dynamics simulations in CHARMM, the effect Hoogsteen conformation has on the entropy of the strand can be examined. The absolute entropy of both Hoogsteen and Watson-Crick conformations can be calculated with the use of a quasi-harmonic approximation using the trajectories of each simulation. Using this method, the entropy contribution of varying amounts of neighboring base pairs can be estimated. This work can be compared with experimental entropy values for entire double-strands. Our calculations show how a localized conformational change can affect the entropy in neighboring site and over longer-range base pairs. 2002-Pos Board B146 Use of Nunchuk Nanostructures for Dynamic dsDNA Bend Angle Measurements by Fluorescence Microscopy Lourdes Velazquez, Deborah Clayton-Warwick, Deborah Fygenson. Physics, UCSB, Goleta, CA, USA. DNA bending is an important factor in the way DNA-binding proteins recognize binding sites and orchestrate transcription or compaction. Current methods for measuring DNA bending (e.g., FRET, X-ray crystallography, cryo-TEM) require specialized training and equipment and offer little or no information on bend stiffness or fluctuations. We introduce a DNA nanostructure tool that makes single-molecule, dynamic measurements of DNA bending accessible to anyone with a research-grade fluorescence videomicroscope. Our tool, nicknamed the ‘‘DNA nunchuk’’, consists of a double stranded DNA linker (~50bp) flanked on either end by DNA nanotubes that are >3um long. We report on calibration of the DNA nunchuk technique using a variety of dsDNA linker sequences including phased poly-A tracts. 2003-Pos Board B147 Dynamic Release of Bending Stress in Short Double-Stranded DNA by Two Types of Deformation CheolHee Kim1, O-Chul Lee1, Jae-Yeol Kim2, Wookyung Sung3, Nam Ki Lee4. 1 Physics, Pohang University of Science and Technology, Pohang, Korea, Republic of, 2Chemical Physics, National Institutes of Health, Bethesda, MD, USA, 3IBS center for Self-assembly and Complexity, Pohang University of Science and Technology, Pohang, Korea, Republic of, 4Physics/School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea, Republic of.
405a
Bending with high curvature is one of the major mechanical properties of double-stranded DNA (dsDNA) for its biological functions. Local-melting in the middle of dsDNA (kink), which reduce the energy cost of bending, have been suggested as alternative DNA conformations in addition to the simple bending of dsDNA in the presence of high constrain force. However, the conformations of deformed dsDNA by high bending force and their dynamic characters remain unknown. Here, we report that the strong bending induces not only the kink in the middle of dsDNA but also the end-melting of dsDNA by applying single-molecule fluorescence resonance energy transfer (smFRET) to D-shaped DNA nanostructure consisted of dsDNA (30 bp) and singlestranded DNA (4 - 30 nt). We directly proved that two deformed structures of dsDNA are not permanent but dynamically interconverted each other in millisecond scales. The transition from end-melting to kink is dominated by entropy (anti-Arrhenius behavior), while the transition from kink to end-melting is dominated by enthalpy. The presence of the mismatch or permanent bubble in dsDNA accelerates the kink formation with less compressive force and the kink state becomes permanent when the size of permanent bubble is larger than three base pairs. 2004-Pos Board B148 Nucleosome Dynamics at Microsecond Timescale: DNA-Protein Interactions, Water-Mediated Interactions and Nucleosome Formation Alexey K. Shaytan1, Grigory A. Armeev2, Alexander Goncearenco1, Victor B. Zhurkin3, David Landsman1, Anna R. Panchenko1. 1 NCBI, National Institutes of Health, Bethesda, MD, USA, 2Faculty of Biology, Lomonosov Moscow State Univeristy, Moscow, Russian Federation, 3Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. At the cornerstone of eukaryotic chromatin is the association of DNA with histone proteins to form nucleosomes. Every nucleosome core particle consists of 147 bp of DNA wrapped around and octamer of histones. Many factors (such as DNA sequence, histone variants, post-translational modifications, etc.) can affect the stability and dynamics of nucleosomes, which in turn provide key mechanisms for epigenetic regulation of gene expression. We report an extensive study of nucleosome dynamics and organization via molecular modeling based on all-atom microsecond molecular dynamics simulations. We analyze dynamics of full nucleosomes including linker DNA segments and fulllength histones in explicit solvent and compare it to the dynamics of nucleosome core particle and histone octamer alone. Extensively long simulation time scale allows us to address the questions of conformation coupling between histones and DNA as well as study the interaction patterns of flexible histone tails. Detailed analysis of protein-DNA interactions is performed including water-mediated interactions and the role of water molecules in the central nucleosome pore. We analyze the rearrangement of nucleosome structure upon DNA binding and discuss the cooperativity of interactions in nucleosome formation. This work was supported by the Intramural Research Programs of NLM and NCI; and RSF grant No. 14-24-00031 (nucleosome visualization algorithms). 2005-Pos Board B149 Effects of Salt on the Stability of a G-Quadruplex from the Human c-MYC Promoter Byul Kim. Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada. In an atmosphere of potassium ions, a modified c-MYC NHE III1 sequence with two G-to-T mutations (MYC22-G14T/G23T) forms a highly stable parallel-stranded G-quadruplex. The G-quadruplex exhibits a steady increase in its melting temperature, TM, with an increase in the concentration of the stabilizing cation Kþ. On the other hand, an increase in the concentration of nonstabilizing Csþ or TMAþ cations at a constant concentration of Kþ causes a sharp decline in TM followed by a leveling off at~200mM Csþ or TMAþ. At 51 C and 600mM Kþ, an increase in Csþ concentration from 0 to 800mM leads to a complete unfolding of the G-quadruplex. These observations are consistent with the picture in which more counterions accumulate in the vicinity of the unfolded state of MYC22-G14T/G23T (nonspecific ion binding) than in that of the G-quadruplex state. We estimate that the unfolded state condenses one extra counterion compared to the G-quadruplex state. Taken together with our earlier results, our data suggest that sodium or potassium cations sequestered inside the central cavity stabilize the G-quadruplex conformation acting as specifically bound ligands. Nonspecifically bound (condensed) counterions may slightly stabilize, exert no influence (human telomeric G-quadruplexes), or strongly destabilize (MYC22-G14T/G23T) the G-quadruplex conformation. We offer a structural rationalization for the enhanced thermal stability of the MYC22-G14T/G23T G-quadruplex.
405a
Bending with high curvature is one of the major mechanical properties of double-stranded DNA (dsDNA) for its biological functions. Local-melting in the middle of dsDNA (kink), which reduce the energy cost of bending, have been suggested as alternative DNA conformations in addition to the simple bending of dsDNA in the presence of high constrain force. However, the conformations of deformed dsDNA by high bending force and their dynamic characters remain unknown. Here, we report that the strong bending induces not only the kink in the middle of dsDNA but also the end-melting of dsDNA by applying single-molecule fluorescence resonance energy transfer (smFRET) to D-shaped DNA nanostructure consisted of dsDNA (30 bp) and singlestranded DNA (4 - 30 nt). We directly proved that two deformed structures of dsDNA are not permanent but dynamically interconverted each other in millisecond scales. The transition from end-melting to kink is dominated by entropy (anti-Arrhenius behavior), while the transition from kink to end-melting is dominated by enthalpy. The presence of the mismatch or permanent bubble in dsDNA accelerates the kink formation with less compressive force and the kink state becomes permanent when the size of permanent bubble is larger than three base pairs. 2004-Pos Board B148 Nucleosome Dynamics at Microsecond Timescale: DNA-Protein Interactions, Water-Mediated Interactions and Nucleosome Formation Alexey K. Shaytan1, Grigory A. Armeev2, Alexander Goncearenco1, Victor B. Zhurkin3, David Landsman1, Anna R. Panchenko1. 1 NCBI, National Institutes of Health, Bethesda, MD, USA, 2Faculty of Biology, Lomonosov Moscow State Univeristy, Moscow, Russian Federation, 3Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. At the cornerstone of eukaryotic chromatin is the association of DNA with histone proteins to form nucleosomes. Every nucleosome core particle consists of 147 bp of DNA wrapped around and octamer of histones. Many factors (such as DNA sequence, histone variants, post-translational modifications, etc.) can affect the stability and dynamics of nucleosomes, which in turn provide key mechanisms for epigenetic regulation of gene expression. We report an extensive study of nucleosome dynamics and organization via molecular modeling based on all-atom microsecond molecular dynamics simulations. We analyze dynamics of full nucleosomes including linker DNA segments and fulllength histones in explicit solvent and compare it to the dynamics of nucleosome core particle and histone octamer alone. Extensively long simulation time scale allows us to address the questions of conformation coupling between histones and DNA as well as study the interaction patterns of flexible histone tails. Detailed analysis of protein-DNA interactions is performed including water-mediated interactions and the role of water molecules in the central nucleosome pore. We analyze the rearrangement of nucleosome structure upon DNA binding and discuss the cooperativity of interactions in nucleosome formation. This work was supported by the Intramural Research Programs of NLM and NCI; and RSF grant No. 14-24-00031 (nucleosome visualization algorithms). 2005-Pos Board B149 Effects of Salt on the Stability of a G-Quadruplex from the Human c-MYC Promoter Byul Kim. Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada. In an atmosphere of potassium ions, a modified c-MYC NHE III1 sequence with two G-to-T mutations (MYC22-G14T/G23T) forms a highly stable parallel-stranded G-quadruplex. The G-quadruplex exhibits a steady increase in its melting temperature, TM, with an increase in the concentration of the stabilizing cation Kþ. On the other hand, an increase in the concentration of nonstabilizing Csþ or TMAþ cations at a constant concentration of Kþ causes a sharp decline in TM followed by a leveling off at~200mM Csþ or TMAþ. At 51 C and 600mM Kþ, an increase in Csþ concentration from 0 to 800mM leads to a complete unfolding of the G-quadruplex. These observations are consistent with the picture in which more counterions accumulate in the vicinity of the unfolded state of MYC22-G14T/G23T (nonspecific ion binding) than in that of the G-quadruplex state. We estimate that the unfolded state condenses one extra counterion compared to the G-quadruplex state. Taken together with our earlier results, our data suggest that sodium or potassium cations sequestered inside the central cavity stabilize the G-quadruplex conformation acting as specifically bound ligands. Nonspecifically bound (condensed) counterions may slightly stabilize, exert no influence (human telomeric G-quadruplexes), or strongly destabilize (MYC22-G14T/G23T) the G-quadruplex conformation. We offer a structural rationalization for the enhanced thermal stability of the MYC22-G14T/G23T G-quadruplex.