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Open Surcelthesis.Pdf The Pennsylvania State University The Graduate School The Huck Institutes of the Life Sciences INTERACTIONS BETWEEN THE COHESIN COMPLEX AND ITS DNA PARTNERS: A STRUCTURAL AND PHYLOGENETIC APPROACH A Thesis in Integrative Biosciences by Alexandra Surcel © 2007 Alexandra Surcel Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2007 The thesis of Alexandra Surcel was reviewed and approved* by the following: Hong Ma Professor of Biology Thesis Advisor Chair of Committee David S. Gilmour Associate Professor of Molecular and Cell Biology William O. Hancock Assistant Professor of Bioengineering Wendy Hanna-Rose Assistant Professor of Molecular and Cell Biology Douglas Koshland Special member Senior staff member of the Carnegie Institution of Washington, Department of Embryology Investigator, Howard Hughes Medical Institute Peter J Hudson Director of the Integrative Biosciences Graduate Degree Program The Huck Institutes of the Life Sciences *Signatures are on file in the Graduate School iii ABSTRACT Cohesin is an evolutionarily conserved protein complex responsible for maintaining sister chromatid cohesion from early S phase to the metaphase-anaphase transition. The cohesin complex is comprised of four proteins – Smc1 and Smc3 that heterodimerize, and Scc1 and Scc3. Imaging of the Smc heterodimer shows that it forms a V shaped molecule and imaging of the entire cohesin holocomplex suggests that it forms an enclosed ring structure. The cohesin complex binds to specific loci along the chromosome arms and centromeres known as Cohesin Attachment Regions (CAR). In lieu of consensus binding sequences at CAR loci, several models have been proposed for cohesin interactions at CAR sites, though no direct structural information about the in vivo interaction of cohesin at CARs has been obtained. This thesis is the first documented effort of the successful isolation and imaging of cohesin-chromatin complexes assembled in vivo. A minichromosome containing a CAR sequence and a CEN3 sequence was isolated from Saccharomyces cerevisiae using the Minichromosome Affinity Purification (MAP) method. The MAP protocol had to be overhauled to obtain high yield from this low-copy centromeric construct. Adjustments to MAP resulted in a 100-fold increase over the yield obtained by using the published protocol. Samples were isolated from G1 synchronized cultures in which cohesin is not bound to CARs and M-phase synchronized cultures in which cohesin is bound to CARs. These MAP isolated samples were negatively and positively stained for TEM analysis. Images showed that replicated minichromosomes always interact with one end of a flexible rod. Length measurements of this protrusion are consistent with a collapsed cohesin ring and width measurements of the rod suggest that multiple cohesins interact at CAR loci. These images lead to a model that suggests conformational changes within the cohesin complex may be responsible for the topological binding of cohesin to chromatin. Phylogenetic analyses were undertaken of the Structural Maintenance of Chromosome (SMC) family of proteins in an effort to identify conserved sequences within the arms of Smc1 and Smc3 that may explain the interaction observed via TEM along the coiled-coil domains of these molecules. Extra disruptions to the coiled-coil iv domains among SMC members were identified. These disruptions further support an alternative to the evolutionary history of these proteins that is presented in phylogenetic trees based solely on sequence alignments. In addition, phylogenetic analysis of the meiotic form of Smc1, known as Smc1β, suggests that it arose via a gene duplication event early in animal divergence, even though it has only been maintained in vertebrate lineages. This study of Smc1β lends support to the evolution of various meiotic regulatory mechanisms among animals. v TABLE OF CONTENTS LIST OF FIGURES......................................................................................................viii LIST OF TABLES .......................................................................................................x LIST OF ABBREVIATIONS.......................................................................................xi Chapter 1 Introduction: Cohesin Structure and Function............................................1 Eukaryotic Cell Cycle ...........................................................................................1 Cohesin structural components .............................................................................3 SMC1 and SMC3...........................................................................................3 SCC1 and SCC3 ............................................................................................6 Cohesin mechanics................................................................................................8 Loading of the mitotic cohesin complex........................................................10 Binding of the mitotic cohesin complex to DNA/chromatin.........................13 Dissolution of the mitotic cohesin complex ..................................................16 Cohesin in meiosis ................................................................................................18 Other proteins essential for cohesin function........................................................18 Other roles for cohesin..........................................................................................20 Overview of thesis ................................................................................................23 Chapter 2 The Construction of CAR-Containing Minichromosomes and the Development of the Minichromosome Affinity Purification (MAP) Technique for Low-Copy Plasmids.......................................................................25 Abstract .................................................................................................................26 Introduction...........................................................................................................27 Results...................................................................................................................30 pCM26-1 plasmid generation and characterization .......................................30 pAS1 plasmid generation and characterization..............................................32 Changes to MAP methodology for pCM26-1................................................36 Additional considerations for the MAP technique.........................................43 Protein analysis of isolated minichromosomes detects presence of cohesin ....................................................................................................43 Discussion and significance ..................................................................................45 MAP methodology.........................................................................................45 Important cis-elements needed for cohesin binding constructs .....................45 Improvements to the MAP technique ............................................................46 Material and Methods ...........................................................................................49 Strains ............................................................................................................49 Plasmids.........................................................................................................49 Cell Synchronization......................................................................................50 Smash and Grab DNA isolation from yeast...................................................50 vi Minichromosome Affinity Purification (MAP).............................................51 LacIZ Column Preparation.............................................................................52 Chromatin Immunoprecipitation (ChIP), PCR, and Data Analysis ...............53 Western blot...................................................................................................53 Chapter 3 Cohesin Interaction at One CAR Locus Shows a Flexible Rod Multi- Complex Structure ................................................................................................54 Abstract .................................................................................................................55 Introduction...........................................................................................................56 Results...................................................................................................................59 TEM analysis from alpha-factor arrested cells shows a singular minichromosome ....................................................................................59 TEM analysis shows a flexible rod protruding from replicated minichromosomes...................................................................................61 Nucleosome mapping at several CAR loci shows well-positioned nucleosomes and no protein footprint.....................................................66 Discussion .............................................................................................................68 Characterization of the pCM26-1 minichromosome .....................................68 Characterization of cohesin bound pCM26-1 and assessment of binding models.....................................................................................................69 Cohesin
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