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A Dissertation Entitled the Study of Protein-Protein Interactions A Dissertation Entitled The Study of Protein-Protein Interactions Involved in Lagging Strand DNA Replication and Repair By Jennifer M. Hinerman Submitted as partial fulfillment of the requirements for the Doctor of Philosophy in Chemistry _______________________________________ Advisor: Dr. Timothy C. Mueser _______________________________________ College of Graduate Studies The University of Toledo August 2008 Copyright © 2008 The document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of The Study of Protein-Protein Interactions Involved in Lagging Strand DNA Replication and Repair Jennifer M. Hinerman Submitted as partial fulfillment of the requirements for the Doctor of Philosophy in Chemistry The University of Toledo August 2008 The organization and coordination of DNA replication machinery at the replication fork is important for accurate, efficient DNA synthesis in all organisms. The initial organization of the replication fork is vital for initiating lagging strand replication, while the regulation of proteins involved in Okazaki fragment processing is important for generating a complete daughter DNA strand. These DNA replication and repair proteins recognize DNA in a structure-specific manner, thus the recognition of these particular DNA structures promote the formation of certain protein-DNA and protein-protein complexes that are essential for DNA replication and repair to occur. Organisms such as the Bacteriophage T4 (T4) and Aeropyrum pernix (Ape) are model systems for use in the study of binary and ternary complexes that form during DNA replication and repair. Reassembling the replication fork would allow the iii determination of the mechanism used to synchronize replication on both the leading and lagging strands. Proteins (helicase assembly protein and single-stranded DNA binding protein) from T4 were used to study the complexes involved in initiation of lagging strand replication. The protein-protein interactions between the helicase assembly protein (59 protein), single-stranded DNA binding protein (32 protein), and truncations of the 32 protein have been investigated (ITC, DSC, DLS, native gels, crystallography). 59 protein had a moderate interaction with 32 protein (KD = 3.7 µM) and with 32-B (KD = 3.6 µM). DNA-protein interactions between the 59 protein and fork DNA substrate (with and without 32 protein) have been studied (fluorescence). X-ray data was collected on a truncation of the 32 protein (32-B). Models of the 59 protein-32-B complex have been elucidated (SAXS, SANS). Ape proteins (proliferating cell nuclear antigen, DNA polymerase B, DNA ligase, and flap endonuclease-1) were characterized to study Okazaki processing. Subunits (Ape0162, Ape0441, and Ape2182) from the heterotrimeric proliferating cell nuclear antigen (PCNA) were cloned, expressed, purified, and characterized (DSC, DLS). DNA ligase was cloned, expressed, and purified. DNA polymerase B has been cloned and expressed. Protein-protein interactions between each of the PCNA subunits, as well as the interactions between each individual subunit and DNA ligase, DNA polymerase B, and flap endonuclease-1 were investigated using mass spectrometry. iv Acknowledgements I would like to thank my advisor, Dr. Mueser (Sir), for the opportunity to work and learn in his lab. I will definitely miss all the “story times”. Thank you for your guidance and for always taking the time to answer all of my questions. I would also like to thank Dr. Hanson for all of his help and guidance. To my fellow lab members, thank you for all the good times, conversations, and friendships. These memories will last forever. I would like to thank my committee members, Dr. Viola, Dr. Funk, and Dr. Lee for all their comments and suggestions. I would like to thank all of my friends, new and old, for their support and friendship. A special thanks goes out to all the new friends I have made at Toledo, especially to the friends whom have made this journey with me. Your friendships helped make the journey more exciting and bearable. To the group of friends whom have supported me as I moved all over the state to go to college and grad school, thank you for everything. Your friendship will always have a special place in my heart. None of this would have been possible without the love and support from my husband, Sam, and family. I could never thank them enough for supporting me through all the good and bad times. No matter how tough things got, they were always there encouraging me the entire way. Their endless support, understanding, and love will always mean the world to me. v Table of Contents List of Figures........................................................................................................................... x List of Tables ........................................................................................................................ xvii List of Abbreviations ............................................................................................................. xix Chapter 1: Introduction............................................................................................................ 1 1.1 Bacteriophage T4 DNA replication ................................................................................4 1.1.1 Helicase assembly protein..................................................................................... 12 1.1.2 DNA helicase protein............................................................................................ 17 1.1.3 Single-stranded DNA binding protein .................................................................. 21 1.1.4 59 protein interactions with 41 helicase and 32 protein ....................................... 25 1.2 Ape lagging strand replication and repair proteins.......................................................29 1.2.1 Proliferating cell nuclear antigen.......................................................................... 32 1.1.2 PCNA - DNA polymerase interactions................................................................. 42 1.2.3 PCNA - Flap Endonuclease 1 interactions............................................................ 46 1.2.4 PCNA – DNA ligase interactions ......................................................................... 48 Chapter 2: Experimental methods........................................................................................... 51 2.1 Vector cloning...............................................................................................................51 2.1.1 Polymerase chain reaction (PCR) ......................................................................... 51 2.1.2 Restriction cloning................................................................................................ 53 2.1.3 Gateway cloning ................................................................................................... 56 2.1.4 Cloning and expression vectors and hosts ............................................................ 58 2.1.5 Preparation of competent cells and transformation protocols............................... 59 2.2 Protein expression and purification ..............................................................................61 vi 2.2.1 Protein expression and lysis.................................................................................. 61 2.2.2 Protein purification ............................................................................................... 63 2.2.3 Protein solubility................................................................................................... 65 2.3 Biophysical studies .......................................................................................................67 2.3.1 Dynamic light scattering....................................................................................... 67 2.3.2 Isothermal titration calorimetry ............................................................................ 68 2.3.3 Differential scanning calorimetry ......................................................................... 69 2.3.4 Fluorescence anisotropy........................................................................................ 70 2.3.5 Small angle X-ray scattering................................................................................. 72 2.3.6 Small angle neutron scattering.............................................................................. 77 2.3.7 X-ray crystallography ........................................................................................... 79 Chapter 3: T-even Bacteriophage DNA replication............................................................... 82 3.1 Bacteriophage T4 results and discussion ......................................................................84 3.1.1 Protein expression and purification ...................................................................... 84 3.1.2 T4 protein characterization ................................................................................... 92 3.1.2.1 Differential scanning calorimetry .................................................................. 92 3.1.2.2 Dynamic light scattering................................................................................ 93 3.1.2.3 Size exclusion chromatography ..................................................................... 95 3.1.2.4 Fluorescence anisotropy................................................................................
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