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Investigating DNA Junction Structure and Dynamics Using a Coarse-Grained Implicit Ion Model for DNA

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Investigating DNA Junction Structure and Dynamics Using a Coarse-Grained Implicit Ion Model for DNA Wesleyan University Physics Department Investigating DNA Junction Structure and Dynamics using a Coarse-grained Implicit Ion Model for DNA by Abraham Kipnis Class of 2019 An honors thesis submitted to the faculty of Wesleyan University in partial fulfillment of the requirements for the Degree of Bachelor of Arts with Departmental Honors in Physics Middletown, Connecticut April, 2019 Abstract Four-way Holliday junctions are cruciform-shaped DNA structures which play vital roles in biological processes. In this thesis, we validate the ability of an explicit ion coarse-grained model for DNA (3SPN.2) to accurately simulate Holliday junction dynamics above and below DNA junction melting temperatures. We analyze a variety of junction behaviors, including ion binding, junction conformations, junction melting, and branch migration, and compare our results with expected results from scientific literature. We discuss four different methods to determine the structure of the junction, evaluate the drawbacks and advantages of these different methods by comparing them with each other and with data from previous studies and show that results qualitatively reflect our expectations of DNA junction structure at equilibrium. Then we use one of these methods to show that DNA junction dynamics as produced by the 3SPN.2 explicit ion model demonstrate the expected trends from literature. Next, we investigate melting by observing the dissociation of individual bases during our simulations and provide quantitative predictions for the dynamics of junction melting. Our results show that melting initiates at specific locations on a per-strand basis. These simulations helped inspire fluorescence melting experiments which strategically place nucleotide base analogs at several locations along junction strands and validate the primary predictions of the modeling. We offer a comprehensive overview of DNA junction research made possible using the 3SPN.2 explicit ion model for DNA and conclude that this model is a useful tool for exploring Holliday junction structure and dynamics in the presence of ions. Acknowledgements I would like to express my utmost gratitude to: My supervisor, Francis Starr, for giving me these projects, providing guidance and inspiration, • patiently helping me hone my skills and find solutions to problems, and reminding me to not lose the forest for the trees. My second supervisor, Ishita Mukerji, for having an eye for details and an intuition that only an • experimentalist can have. Other members of the Mukerji lab, including Rachel Savage, Julie McDonald, Nick Taylor, and • Dacheng Zhao, for their contributions to experimental four-way junction research. Members of the Wesleyan physics department: Tsampikos Kottos, Greg Voth, Renee Sher, Fred • Ellis, Tom Morgan and Yunseong Nam, for teaching me more than I could have imagined during my time here. Other current and ex members of the Starr lab, including Wujie Wang, Xinyu Zhu, Natalie • Strassheim, Abhishek Fakiraswami, Wenghang Zhang, Hamed Emamy, Amber Storey, Chloe Thorburn and Nathan Shankman for laying the initial groundwork this project and for sharing expertise, advice, and being my personal heroes. The residents of 59 Warwick: Will Barr, Cail Daley, Ryan Adler-Levine, Gabe Weinreb, and Tony • Strack for being with me during the summer of 2017. My family, for providing love and care the whole way through. • ii Contents 1 Introduction 1 1.1 Homologous Recombination . .2 1.2 DNA Junction Studies . .6 1.3 DNA Junction Melting . 12 1.4 DNA Nanotechnology . 14 1.5 Motivation and Objectives . 16 2 Model and Simulations 17 2.1 Coarse-grained DNA Model . 18 2.2 Molecular Simulations . 20 2.3 Hidden Markov models and our implementation . 23 3 Equilibrium Structure and Dynamics 25 3.1 Radial distribution function . 25 3.2 Methods to determine junction isoform . 28 3.2.1 Central base distances . 28 3.2.2 Consecutive phosphate angles . 31 3.2.3 RMSD between ideal isoform structures and current snapshot . 33 3.2.4 Markov model state classification . 34 3.3 Transitions between conformations . 38 iii CONTENTS CONTENTS 4 Melting Dynamics 39 4.1 Method to determine base dissociation . 40 4.2 Preferential melting . 42 4.3 Isoform transitions during melting process . 44 5 Branch Migration Dynamics 47 5.1 Methods to determine junction location . 48 5.2 Junction migration probability . 48 6 Conclusion 51 6.1 Evaluation of ionic distributions around the junction . 51 6.2 Evaluation of junction isoform determination methods . 52 6.3 Evaluation of melting data . 53 6.4 Future directions . 54 iv List of Figures 1.1 Meiotic recombination . .2 1.2 Holliday model . .3 1.3 Junction conformation and branch migration energy landscape . .4 1.4 Holliday junction formation and branch migration . .5 1.5 Holliday junction resolution . .5 1.6 Products from cleavage of junction by restriction enzymes . .6 1.7 Electrophoretic gel mobilities of restriction enzyme products . .7 1.8 DNA junction conformer transitions . .8 1.9 FRET experiment to determine conformer populations as a function of [Mg2+].....9 1.12 Simulations of B-DNA show increased flexibility at high salt concentrations . 10 1.10 FRET experiment to determine conformer populations as a function of other ions . 11 1.11 FRET experiments of mobile junctions to determine junction migration rates . 11 1.13 Radial distribution function between Mg2+ions and DNA junction phosphates . 12 1.14 Junction melting curve from implicit ion model for DNA . 13 1.15 Two-dimensional lattices made from DNA junction subunits . 14 1.16 Nanotubes made from DNA junction subunits . 15 2.1 Illustration of coarse-grained representation of B-DNA using 3SPN.2 model . 18 2.2 Fraction of broken bases from melting simulations . 22 3.1 Sodium radial distribution functions . 26 3.2 Magnesium radial distribution functions. 26 v LIST OF FIGURES LIST OF FIGURES 3.3 Schematic of isoform determination using base distances . 28 3.4 Implicit ion model central base distance histograms and isoform population distributions 29 3.5 Explicit ion central base distance histogram . 30 3.6 Isoform population distributions from base distances criterion using explicit ion model . 30 3.7 Angle between consecutive phosphates . 31 3.8 Explicit ion central phosphate angle histograms . 32 3.9 Isoform population distributions from central phosphate angles criterion . 32 3.10 Isoform population distribution by using RMSD from ideal isoform . 33 3.11 Isoform population distributions from base distances Markov models . 35 3.12 Isoform population distributions from phosphate angles Markov models . 36 3.13 Isoform population distributions from base distances and angles Markov models . 37 3.14 Transition matrices from phosphate angles criterion . 38 4.1 Stages of the melting process . 39 4.2 Example of base pair distances and melting time . 41 4.3 Number of base pairs and tm from one melting simulation . 41 4.4 Mean base pair dissociation time tm at 360K . 43 4.5 Experimental fluorescence melting curves . 44 4.6 Theoretical melting temperatures for individual strands that comprise the J3 junction . 45 4.7 Isoform timeseries for a single junction melting simulation . 46 4.8 Isoform population distribution during melting . 46 5.1 Junction location timeseries, [Na+] 100mM [Mg2+] 0mM . 49 5.2 Junction location timeseries, [Na+] 100mM [Mg2+] 50mM . 49 5.3 Junction migration probability . 50 vi Chapter 1 Introduction Science, for me, gives a partial explanation for life. “ In so far as it goes, it is based on fact, experience and experiment. ” Rosalind Franklin, a letter to Ellis Franklin, 1940 DNA is an information-encoding polymer responsible for carrying instructions for how organisms live, procreate, and die. For solving the most common structure of DNA, the method by which DNA replicates, Watson, Crick, and Wilkins won the Nobel Prize for Physiology in 1962 with the help of x-ray diffraction data from Rosalind Franklin [1]. DNA is composed of two oppositely oriented strands of single-stranded DNA (ssDNA) in a right-handed helix, with bases on opposing strands connected through hydrogen bonding. Under physiological conditions, double-stranded DNA (dsDNA) exists in “B form” (B-DNA), with one turn of the helix approximately every 10 base pairs, or 34 A˚ . Most of the time, DNA is organized into condensed networks called chromosomes. In cells, helicases and polymerases aid DNA replication by unzipping the strands and using each as a template for synthesis of a new complementary strand. Imperfect copying and mismatch repair results in approximately one point mutation occurring per 10 billion bases replicated [2], which can alter protein structure and function. A diversity of phenotypes within a population contribute to a species’ ability to withstand environmental perturbations [2]. 1 Section 1.1 - Homologous Recombination 2 1.1 Homologous Recombination Another important evolutionary tool that increases genetic diversity is meiotic recombination. During meiosis (the repeated division of cells into four cells with half the number of chromosomes), duplicated homologous chromosomes align at the centers of cell nuclei before segregating and triggering cell division [See Figure 1.1]. Recombination begins between aligned homologous chromosomes at sites called chiasmata when recombination proteins create two “nicks” in similar regions of a single strand on each chromosome [3]. Figure 1.1: A biological pathway in which DNA junctions appear. In fungi, plants, and male mammals, meiosis begins with the duplication of the cell’s chromosomes (a). Meiotic recombination is then carried out by the formation and resolution of Holliday junctions (b). To complete meiosis, the cell divides into two cells with two sets of chromosomes each (c), and then division occurs again as each of those cells divides into one cell with one set of chromosomes each (d). An enzyme complexes with the nicked strands, separating them from their intact antisense strand. This enzyme-strand complex then searches for a similar sequence on the opposing chromosome, and the nicked strands cross over and pair with the antisense strand of the homologous duplex.
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