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Characterizing Excluded Strand DNA Interactions with Hexameric Helicases and Determining Roles in Unwinding Mechanisms by Sean M

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Characterizing Excluded Strand DNA Interactions with Hexameric Helicases and Determining Roles in Unwinding Mechanisms by Sean M Characterizing Excluded Strand DNA Interactions with Hexameric Helicases and Determining Roles in Unwinding Mechanisms by Sean M. Carney Bachelor of Science, Gwynedd-Mercy University, 2011 Submitted to the Graduate Faculty of the Kenneth P. Dietrich School of Arts and Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2016 UNIVERSITY OF PITTSBURGH DIETRICH SCHOOL OF ARTS AND SCIENCES This dissertation was presented by Sean M. Carney It was defended on July 14th, 2016 and approved by Andrea J. Berman, Ph.D., Assistant Professor, Biological Sciences Saleem A. Khan, Ph.D., Professor, Microbiology and Molecular Genetics Patricia L. Opresko, Ph.D., Associate Professor, Environmental and Occupational Health Dissertation Advisor: Sanford H. Leuba, Ph.D., Associate Professor, Cell Biology Dissertation Advisor: Michael A. Trakselis, Ph.D., Associate Professor, Chemistry and Biochemistry, Baylor University ii Copyright © by Sean M. Carney 2016 iii Characterizing Excluded Strand DNA Interactions with Hexameric Helicases and Determining Roles in Unwinding Mechanisms Sean M. Carney, PhD University of Pittsburgh, 2016 DNA replication is an essential process for all living organisms, and errors in this process can lead to genetic mutations and disease. An assembly of protein machinery, termed the replisome, coordinates enzymatic activities at the replication fork. The DNA helicase is the heart of the replisome, unwinding double-strand DNA at the head of the progressing replisome and providing single-strand templates for DNA polymerases. Replicative helicases are composed of six subunits, and arranged in a ring-like structure where ATP hydrolysis events provide the energy to translocate upon and unwind the DNA. The mechanism of helicase unwinding has been widely studied, but there are still many aspects that remain unknown. It is generally thought that these helicases encircle one strand of DNA while the other is excluded from the central channel of the helicase. Our lab has previously identified an interaction between the excluded strand and the helicase exterior that was important for unwinding in the archaeal MCM helicase. The steric exclusion model of replicative helicase unwinding was expanded to include the excluded strand interactions in this newly proposed steric exclusion and wrapping (SEW) model. Here, we present work that expands on the SEW model by revealing that the bacterial DnaB and mitochondrial Twinkle replicative helicases also interact with the excluded strand. We have also developed a new single-molecule FRET analysis program to characterize these excluded strand interactions. Although the excluded strand interaction is seen in multiple replicative helicases, we propose distinct roles for the interaction based on functional assays and known differences in replisome architecture across the various organisms. We have also begun to characterize the iv helicase-excluded strand wrapping interaction in the presence of other replisome components, namely the single-strand binding (SSB) protein. We further characterized the archaeal SSB protein from Sulfolobus solfataricus and provide evidence for a novel DNA-helicase-SSB ternary complex. Overall, this thesis makes significant contributions to the understanding of replicative helicase unwinding mechanisms by expanding upon the current steric exclusion and wrapping model and introduces a novel single-molecule FRET analysis program that we anticipate will be adopted and utilized by others in the field. v TABLE OF CONTENTS PREFACE ................................................................................................................................. XVI 1.0 INTRODUCTION ........................................................................................................ 1 1.1 DNA REPLICATION ......................................................................................... 1 1.2 DNA HELICASES ............................................................................................... 5 1.2.1 Helicase Core Domains: RecA-like and AAA+ .............................................. 6 1.2.2 Superfamilies 1-2 ............................................................................................. 8 1.2.3 Superfamilies 3-6: The Hexameric Helicases .............................................. 10 1.3 HEXAMERIC HELICASE UNWINDING: MECHANISMS AND MODELS ............................................................................................................................. 11 1.3.1 Hexameric Helicase Structure ...................................................................... 11 1.3.2 Hexameric NTPase Activity .......................................................................... 13 1.3.3 Hexameric Helicase Unwinding Models ...................................................... 15 1.3.4 Impact and Applications ............................................................................... 17 1.4 THE EXCLUDED STRAND INTERACTION: DISCUSSING IN VIVO ROLES ............................................................................................................................. 18 1.4.1 The Steric Exclusion and Wrapping (SEW) Model of Unwinding. .......... 18 1.4.2 Impact of the Excluded Strand in Unwinding ............................................ 20 1.4.2.1 Excluded Strand as a ‘Molecular Ratchet’ ....................................... 20 vi 1.4.2.2 Excluded Strand as a ‘Molecular Brake’ .......................................... 20 1.4.2.3 Nonhexameric Helicases that Engage Both Strands ........................ 21 1.4.3 Sensing DNA Damage ................................................................................... 24 1.4.4 Role of the Excluded Strand in the Unwindosome ..................................... 26 1.4.5 Conclusion ...................................................................................................... 30 2.0 EXCLUDED STRAND WRAPPING REGUALTES DNAB HELICASE ACTIVITY................................................................................................................................... 31 2.1 SUMMARY ........................................................................................................ 31 2.2 INTRODUCTION ............................................................................................. 32 2.3 MATERIALS AND METHODS ...................................................................... 34 2.3.1 Materials ......................................................................................................... 34 2.3.2 Cloning and Purification of EcDnaB ........................................................... 35 2.3.3 Purification of EcDnaC ................................................................................. 36 2.3.4 Single-Molecule Fluorescence Resonance Energy Transfer ...................... 37 2.3.5 Single-Molecule FRET Data Analysis and ExPRT Plots ........................... 38 2.3.6 EcDnaB Structural Homology Model .......................................................... 39 2.3.7 Gel Based Helicase Unwinding Assays ........................................................ 39 2.3.8 ATPase Assay ................................................................................................. 40 2.3.9 Fluorescence Anisotropy ............................................................................... 40 2.3.10 ssDNA Translocation Assays ....................................................................... 41 2.3.11 Fluorescence DNA Unwinding Assays ........................................................ 42 2.4 RESULTS ........................................................................................................... 43 2.4.1 Developing Methods to Characterize Excluded Strand Interactions ....... 43 vii 2.4.1.1 Single-Molecule Fluorescence Energy Transfer ............................... 44 2.4.1.2 Explicit Probability and Rate Transition (ExPRT) Plots. ............... 47 2.4.2 EcDnaB Interacts with the Excluded Strand .............................................. 51 2.4.3 EcDnaB and SsoMCM Wrap the Excluded Strand Similarly .................. 54 2.4.4 Conservered Surface Mutants of EcDnaB Alter Excluded Strand Wrapping .................................................................................................................... 60 2.4.5 Surface Mutants Enhance DNA Unwinding Activity ................................. 66 2.4.6 Basis of K180A and R328A/R329A Unwinding Hyperactivity ................. 69 2.4.7 ssDNA Translocation of Surface Mutants ................................................... 72 2.5 DISCUSSION ..................................................................................................... 82 2.5.1 EcDnaB and SsoMCM Interact Similarly with the Excluded Strand ...... 82 2.5.2 Probing EcDnaB Excluded Strand Binding Path via Mutational Analysis . ......................................................................................................................... 83 2.5.3 Disrupting Excluded Strand Interactions Stimulates EcDnaB Unwinding . ......................................................................................................................... 84 2.5.4 Comparison with Previous Studies on EcDnaB Unwinding Mechanism . 86 2.5.5 Potential In Vivo Roles of Excluded Strand Interactions .......................... 87 2.6 ACKNOWLEDGEMENTS .............................................................................. 88 2.7 FUNDING ..........................................................................................................
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