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STRUCTURAL and FUNCTIONAL STUDIES of the BACTERIAL RECA PROTEIN DISSERTATION Presented in Partial Fulfillment of the Requirement

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STRUCTURAL and FUNCTIONAL STUDIES of the BACTERIAL RECA PROTEIN DISSERTATION Presented in Partial Fulfillment of the Requirement STRUCTURAL AND FUNCTIONAL STUDIES OF THE BACTERIAL RECA PROTEIN DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Rakhi Rajan, M.S. * * * * * The Ohio State University 2007 Dissertation Committee: Dr. Charles E. Bell, Advisor Approved by Dr. Dehua Pei Dr. Scott Walsh Dr. Ross Dalbey Advisor Biophysics Graduate Program ABSTRACT Double stranded (ds) DNA breaks are among the most detrimental types of DNA damage. dsDNA breaks can be repaired in cells by a process called homologous recombination. RecA is the key player that mediates the DNA strand exchange reaction in the recombination process. The gram positive bacterium Deinococcus radiodurans (Dr) is extremely resistant to high doses of ionizing radiation and thus of great interest for studying biological DNA repair processes and is of potential use in the bioremediation of radioactive waste. The resistance of Dr to extreme doses of ionizing radiation depends on its highly efficient capacity to repair dsDNA breaks. The Dr RecA protein promotes DNA strand-exchange by an unprecedented inverse pathway, in which the presynaptic filament is formed on dsDNA instead of ssDNA. In order to gain insight into the remarkable DNA repair capacity of Dr and the novel mechanistic features of its RecA protein, the x-ray crystal structure of Dr RecA in complex with ATPγS was determined at 2.5Å resolution. Like RecA from E. coli, Dr RecA crystallizes as a helical filament that is closely related to its biologically relevant form, but with a more compressed pitch of 67Å. Although the overall fold of Dr RecA is similar to E. coli RecA, there is a large reorientation of the C-terminal domain, which in E. coli RecA has a site for binding dsDNA. Compared to E. coli RecA, the inner surface along the central axis of the Dr RecA filament has an increased positive electrostatic potential. Unique amino acid ii residues in Dr RecA cluster around a flexible β-hairpin that has also been implicated in DNA binding. The details of Dr RecA structure are discussed in chapter 2. RecA generally binds to any sequence of ssDNA but has a preference for GT-rich sequences, as found in the recombination hot spot Chi (5’-GCTGGTGG-3’). When this sequence is located within an oligonucleotide, binding of RecA is phased relative to it, with a periodicity of three nucleotides. This implies that there are three separate nucleotide-binding sites within a RecA monomer that may exhibit preferences for the four different nucleotides. In chapter 3, a RecA coprotease assay was used to further probe the ssDNA sequence specificity of E. coli RecA protein. The extent of self- cleavage of a λ-repressor protein fragment in the presence of RecA, ADP-AlF4, and 64 different trinucleotide-repeating 15-mer oligonucleotides was determined. The coprotease activity of RecA is strongly dependent on the ssDNA sequence, with TGG-repeating sequences giving by far the highest coprotease activity, and GC and AT-rich sequences the lowest. For selected trinucleotide-repeating sequences, the DNA-dependent ATPase and DNA-binding activities of RecA were also determined. The DNA-binding and coprotease activities of RecA have the same sequence dependence, which is essentially opposite to that of the ATPase activity of RecA. The implications with regard to the biological mechanism of RecA are discussed. The inverse strand exchange pathway of Dr RecA was proposed based on in vitro strand exchange reactions, which gives an indirect measurement of the RecA-DNA iii interaction. The crystal structure of Dr RecA showed features consistent with the inverse strand exchange mechanism. In chapter 4, a set of experiments was designed to directly measure the interactions of Dr and Ec RecA proteins with ssDNA and dsDNA substrates. The experiments do not reveal any distinctive differences in the DNA-binding properties of the two proteins that are consistent with the proposed model for the inverse strand exchange pathway of Dr RecA. Chapter 5 summarizes the work done in collaboration with Dr. Pei. S- Ribosylhomocysteinase (LuxS) is the enzyme which catalyses the synthesis of the precursor of type II bacterial quorum sensing molecule (AI-2). AI-2 is very important in antibiotic development, since it mediates inter-species communication with in bacteria. A catalytically inactive mutant (C84A) of Bacillus subtilis LuxS (BsLuxS) was co- crystallized with the 2-ketone intermediate and the structure was determined to 1.8 Å resolution. The structure reveals that the C2 carbonyl oxygen is directly coordinated with the metal ion, providing strong support for the proposed Lewis acid function of the metal ion during catalysis. A series of structural analogues of the substrate for LuxS were designed and synthesized. Co-crystal structures of the wild type BsLuxS bound with two of these compounds largely confirmed the design principles, i.e., the importance of both the homocysteine and ribose moieties in the high-affinity binding to LuxS active site. iv Dedicated to my family v ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Charles E. Bell, for his support and guidance throughout my graduate education. His guidance and insight were instrumental in the success of the research presented in this dissertation. I am grateful to my committee members Dr. Dehua Pei, Dr. Ross Dalbey, and Dr. Scott Walsh for their valuable suggestions in the preparation of the thesis. I am thankful to Dr. Russ Hille for serving in the committee for a major period and also for his inputs in developing the dissertation. Special thanks are due to Dr. Pei and his lab members, especially Dr. Zhu, who were involved in part of the work presented in this dissertation. I would like to thank Dr. Scott Walsh for access to the BIAcore instrument and also for his guidance in conducting and interpreting the SPR experiments. I also thank Dr. Hille for the use of the gel documentation system in his laboratory. I am thankful to Dr. Kalpana Ghoshal and Dr. Sarmila Majumder for help with the gel extraction and purification techniques. I am indebted to Dr. Bell’s lab members Jinjin, Xu, Jim, and Dr. Ndjonka for the stimulating discussions and help throughout the research. vi I am grateful to the Ohio State Biophysics Graduate Studies Program and Dr. Thomas Clanton for accepting me into the Program. I extend my special thanks to Susan Hauser for all her help during the graduate studies. I also thank the Department of Molecular and Cellular Biochemistry, and the administrative members, Barbara Nesbitt, Brenda Blanton, Ron Louters, and Eric Robbins for all their help during the course of the study. I extend very special thanks to my husband Sudarshan Seshadri who provided moral support and encouragement throughout my doctoral work. I am proud of my daughter Meena for surviving through the difficult times throughout my graduate studies. Finally, I would like to thank my parents, Rajasekharan Nair and Leelamony, and my sister Reshmi, who provided the strong foundation for all the achievements in my life. This research was supported by the funds available to Dr. Charles E. Bell from the ‘National Institute of Health’ and to research collaborator Dr. Dehua Pei from ‘National Institute of Health’. vii VITA May, 15, 1976 ................................................................................Born- Trivandrum, India 1994-1998 ...................... B.Sc. Agriculture, Kerala Agricultural University, Kerala, India 1998-2000 M.Sc. Biotechnology, Tamil Nadu Agricultural University, Coimbatore, India 2000-2002 ..............Junior Research Fellow, Madurai Kamaraj University, Madurai, India 2002-present...Graduate Research Associate, The Ohio State University, Columbus, USA PUBLICATIONS 1. Rajan, R., and Bell, C. E. (2004). Crystal structure of RecA from Deinococcus radiodurans: insights into the structural basis of extreme radioresistance. J. Mol. Biol. 344, 951-963. 2. Rajan, R., Zhu, J., Hu, X., Pei, D., and Bell, C. E. (2005). Crystal structure of S- ribosylhomocyteinase (LuxS) in complex with a catalytic 2-ketone intermediate. Biochemistry 44, 3745-3753. 3. Rajan, R., Wisler, J. W., and Bell, C. E. (2006). Probing the DNA sequence specificity of Escherichia coli RECA protein. Nucleic Acids Res. 34, 2463-2471. 4. Shen, G., Rajan, R., Zhu, J., Bell, C. E., and Pei, D. (2006). Design and synthesis of substrate and intermediate analogue inhibitors of S-ribosylhomocysteinase. J. Med. Chem. 49, 3003-3011. viii FIELDS OF STUDY Major Field: Biophysics ix TABLE OF CONTENTS Page Abstract............................................................................................................................... ii Dedication........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita ..................................................................................................................................viii List of tables..................................................................................................................... xix List of figures.................................................................................................................... xx Abbreviations................................................................................................................. xxiv Chapters: 1. Introduction
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