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AFM Investigations of Critical Interactions in the Bacillus Primosome and Cryogenic AFM a New Tool for Structural Biology

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AFM Investigations of Critical Interactions in the Bacillus Primosome and Cryogenic AFM a New Tool for Structural Biology AFM investigations of critical interactions in the Bacillus primosome and Cryogenic AFM a new tool for structural biology Ian James Turner BSc (Hons), MSc Thesis submitted to The University of Nottingham for the degree of Doctor of Philosophy September 2005 This thesis is dedicated to my mum and dad; I know what you have been through to get me here. 2 Abstract In this thesis for the first AFM has been employed for the high resolution imaging of a protein assembly. The DnaB-DnaG Helicase-Primase interaction in Bacillus is the key reaction that causes the switch from primase mode to polymerisation mode. This assembly was imaged using the AFM to a sub- molecular resolution revealing structural detail of the interaction. It is shown that the binding of the primase causes the structure of the helicase to switch from a hexamer to a trimer of dimers with one primase molecule bound to each dimer; also the existance of sub-populations with one and two primases bound suggests a sequential mode of binding. Recently crystallography data has been published that confirms the structural observations generated by AFM here. This is the first time that AFM and crystallography data have been used concurrently to solve the molecular structure of a protein assembly and it shows the potential application of AFM for sub-molecular resolution imaging of other protein assemblies. The role of DnaD in the Bacillus primosome is well established, however, its exact function was unknown. In this thesis AFM was applied to help solve this biomolecular problem, it revealed that DnaD has a pivotal role in early primosome assembly, ‘opening up’ the DNA allowing other components of the cascade to bind. DnaD was shown to cause supercoiled DNA to adopt an open circular formation; this reaction was shown to be both reversible and universally applicable to all sequences of DNA. Comparisons are made between the role of DnaD and the roles of the histone-like proteins H-NS and HU. These experiments show that AFM can be applied to the imaging of proteins and their interactions with DNA and used to solve biomolecular problems that other techniques cannot solve. The design and implementation of a novel cryogenic AFM system for the imaging of biomolecules at subzero temperatures was executed. Preliminary results show that such a system has the potential to reduce the two main intrinsic effects limiting current AFM imaging; sample softness and thermal motion. 3 The application of AFM in this thesis shows its strength as a tool in molecular biology not only for the high resolution imaging of proteins and protein assemblies but also as a technique that can be uniquely applied to solve biomolecular problems. This thesis also shows for the first time that AFM can be applied to generate sub-molecular resolution of protein assemblies. The strength of the AFM data when combined with crystallography data shows that AFM is a very powerful tool for the imaging of protein assemblies; it could even become the technique of choice. 4 Table of Contents Abstract….........................................................................................3 Contents….........................................................................................5 List of Figures…..............................................................................11 List of Tables…...............................................................................17 List of Abbreviations……………………………………………………..18 1. Introduction….............................................................................19 1.1 Background…......................................................................19 1.2 Modes of operation….............................................................21 1.3 Additional signals…...............................................................24 1.4 Limitations…........................................................................24 2. Bacillus….....................................................................................26 2.1General….............................................................................26 2.1.1 Endospore….................................................................27 2.1.2 Species…......................................................................29 2.2. Replication Overview…........................................................30 2.3. Replication Initiation….........................................................32 2.3.1 Initiation regulation….....................................................33 2.3.2 The primosomal cascade…...............................................35 3. Methods…...................................................................................39 3.1 AFM Imaging…...................................................................39 3.1.1 Air Imaging….................................................................39 3.1.2 Liquid Imaging…............................................................40 3.2 DNA preparation…...............................................................40 5 3.2.1 Harvesting bacteria susceptible for transfection. …..............40 3.2.2. Transforming the bacteria…............................................40 3.2.3. Removing non-transformed bacteria…...............................41 3.2.4. Purifying plasmid…........................................................41 3.2.5 Check for plasmid DNA….................................................41 3.2.6 Restriction digest…..........................................................43 4. The interaction of the bacterial helicase (DnaB) and primase (DnaG) 4.1. Introduction…......................................................................43 4.1.1 General….......................................................................43 4.1.2 Helicases….....................................................................43 4.1.3 DnaB Helicase….............................................................47 4.1.4 DnaG Primase….............................................................49 4.1.5 DnaBG Complex…..........................................................50 4.2. Method …..........................................................................52 4.2.1 Atomic Force Microscopy…..............................................52 4.2.2 Protein Purification…......................................................52 4.2.3 BG Complex…................................................................52 4.2.4 Complementary Techniques…...........................................53 Site-directed mutagenesis …..............................................53 Analytical gel filtration…..................................................53 ATPase assays….............................................................53 Helicase assays…............................................................54 Yeast two-hybrid (Y2H) experiments…................................54 Limited proteolysis….......................................................55 4.3. Results…...............................................................................56 4.3.1 DnaB Helicase….............................................................56 4.3.2 DnaG primase…..............................................................58 6 4.3.3 DnaBG….........................................................................59 4.3.4 Subpopulations….............................................................61 4.4. Complementary Work….......................................................63 4.4.1 Mutations in the N-Domain and n linker region of DnaB…....64 4.4.2 Mutations in the C-domain of DnaB…................................65 4.4.3 The N- & C-domains of DnaB do not interact with DnaG…...65 4.4.4 Primase-mediated protection of the linker region…..............65 4.5. Discussion….........................................................................67 4.6. Recent Advances…..............................................................70 5.What is the role of DnaD in the Bacillus subtilis Primosome? 5.1. Introduction…......................................................................73 5.2. Method…...........................................................................79 5.2.1 Atomic Force Microscopy…..............................................79 5.2.2 DnaD and pBR322 preparation…......................................79 5.2.3 Complementary techniques…............................................79 5.2.3.1 Protein purifications…........................................80 5.2.3.2 Sedimentation velocity experiments…………………..81 5.2.3.3 Gel shift assays…...............................................81 5.2.3.4 Analytical gel filtration….....................................82 5.3. Results…..............................................................................83 5.3.1 Imaging of DnaD Protein…..............................................83 5.3.2 Imaging of supercoiled pBR322…......................................87 5.3.3 Imaging of DNA and DnaD Protein complexes…..................88 5.3.4 DnaD-DNA Complexes in Liquid…....................................91 5.4. Complementary Data….........................................................95 5.4.1 DnaD Oligomeric State….................................................95 5.4.2 DnaD Mediated Nucleoprotein Complexes….......................97 5.5. Discussion….........................................................................99 7 5.6. Model…...............................................................................105 6. Is DnaD a global regulator of DNA architecture?
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