EXTRACELLULAR NUCLEASE FROM BASIDIOBOLUS HAPTOSPORUS (89-3-24) : PURIFICATION AND CHARACTERIZATION A THESIS SUBMITTED TO THE UNIVERSITY OF PUNE FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY BY NEELAM A. DESAI DIVISION OF BIOCHEMICAL SCIENCES NATIONAL CHEMICAL LABORATORY PUNE 411 008 (INDIA) NOVEMBER 2000 DECLARATION Certified that the work incorporated in the thesis entitled "Extracellular nuclease from Basidiobolus haptosporus (89-3-24) : Purification and characterization" submitted by Miss. Neelam A. Desai was carried out under my supervision. Such material as has been obtained from other sources has been duly acknowledged in the thesis. Dr. V. Shankar Research Guide i ACKNOWLEDGMENT I take this opportunity to gratefully acknowledge my guide Dr. V. Shankar for his guidance and keen interest during the course of this investigation. I am equally obliged to Dr. M. I. Khan for his valuable suggestions, which proved to be turning points in the successful completion of this work. I would also like to extend my sincere thanks to : Dr. M. C. Srinivasan, emeritus scientist, Division of Biochemical Sciences, NCL, for his kind gift of the culture Basidiobolus haptosporus (89-3- 24). It was because of his interest in the microbial world and urge to isolate new strains that I had an opportunity to learn many interesting aspects of the different microbial strains isolated by him. Dr. K. N. Ganesh, Head, Organic Chemistry (S), NCL, for providing synthetic deoxyribooligonucleotides and for permission to use HPLC and CD facilities. Dr. S. K. Rawal, Division of Plant Tissue Culture, NCL, for HPLC and lyophilisation facilities. Dr. (Mrs.) J.N. Nandi, Dr. D.T. Mourya and Mr. Pradeep, National Institute of Virology, Pune, for their kind co-operation and help in conducting FPLC, protein blotting and glycoprotein staining experiments. Dr. Dinkar Salunke, National Institute of Immunology, New Delhi, for N-terminal sequencing. Dr. C.G. Suresh, Dr. (Miss) Aditi Pant, Dr. M.V. Deshpande, Dr. (Mrs.) M. V. Rele, Dr. (Mrs.) H. Sivaraman and Dr. (Mrs.) Vidya Gupta, Division of Biochemical Sciences, NCL, and Dr. Krishna Sastry, National Center for Cell Sciences, Pune, for their timely help. Mr. Modak, Mr. Kamthe and Mr. Karanjkar for efficient maintenance of equipments. ii Head, Division of Biochemical Sciences, for his encouragement and support. Director, NCL, for permitting me to submit this work in the form of a thesis and Council of Scientific and Industrial Research, India, for financial assistance. A number of colleagues and friends have always extended their helping hand at various stages during the course of this work. I would like to specially thank Rao, Ramchander, Vrinda, Bhushan, Mukund, Anjali and Pradeep for their help. It was indeed fortunate to have Mrs. Sreedevi U. as my room partner whose caring and helpful nature made my stay in the hostel comfortable and memorable. Discussions with my lab mates Rangarajan, Rohini and Rajashree have always helped in critically evaluating my work. I am thankful to them for their support and co-operation. The atmosphere in the lab was always cheerful because of Dr. (Mrs.) Sumedha Deshmukh. I would like to specially thank Mr. and Mrs. Deshpande and Nagnath who despite their busy schedule, always went out of their way to help me. I appreciate the helpful nature of Ashish, Anand and Vineet. My mother has been a source of inspiration and the driving force during all these years. No words can suffice to acknowledge the tremendous moral support and sacrifices of my parents. It was because of the love and blessings of my family that I could successfully complete this work. All this would have been impossible without the strong emotional support and co-operation by my husband Dr. Subray Hegde, who has incessantly helped me through all the ups and downs during the course of this investigation. Finally, I would like to thankfully acknowledge Professor M.V. Hegde, for his interest in my work, encouragement and timely suggestions. Neelam A. Desai. iii iv DEDICATED AFFECTIONATELY TO MY PARENTS CONTENTS Page No. DECLARATION i ACKNOWLEDGEMENTS ii SUMMARY 1 - 5 Chapter 1 : General Introduction 6 - 75 Single-strand-specific nucleases 6 Historical perspectives 6 Occurence and localization 7 Assay 9 Detection 11 Purification 12 Physicochemical properties 14 Optimum pH and pH stability 14 Optimum temperature and temperature stability 15 Metal ion requirement 16 Effect of salt concentration 18 Stability to denaturants 19 Effect of organic solvents 19 Inducers, activators and inhibitors 20 Molecular mass and subunit structure 23 Isoelectric point 26 Glycoprotein nature 27 Substrate specificity 28 Mode of action 30 Conformation specificity 33 Action on polynucleotides 33 Action on supercoiled and covalenlty closed DNA 35 Action on oligonucleotides that give rise to single -stranded loops 40 Base / linkage specificity 44 Action on modified substrates 48 Structure specific DNA cleavage 52 Associated phosphomonoesterase activity 54 Structure and function 55 Coordination and function of metal ions 57 Substrate binding and catalysis 60 Water assisted metal ion catalysis 62 Chemical nucleases 65 Biological role 66 Applications 70 Present investigation 75 Chapter 2 : Purification and characterization of the deoxyribonuclease activity. 76 - 123 Summary 76 Introduction 76 Materials 77 Methods 78 Results and discussion 90 Conclusion 123 Chapter 3 : Characterization of the associated 124 - 140 ribonuclease activity. Summary 124 Introduction 124 Materials 125 Methods 125 Results and discussion 127 Conclusion 140 Chapter 4 : Characterization of the associated 141 - 158 3'-phosphomonoesterase activity. Summary 141 Introduction 141 Materials 142 Methods 142 Results and discussion 144 Conclusion 158 Chapter 5 : Active site characterization. 159 - 170 Summary 159 Introduction 159 Materials 160 Methods 160 Results and discussion 164 Conclusion 170 General Discussion 171 - 173 REFERENCES 174 - 198 CHAPTER 1 GENERAL INTRODUCTION SINGLE-STRAND-SPECIFIC NUCLEASES Historical perspectives Nucleic acids act as carriers of genetic information from one generation to the other. In order that the genetic information is faithfully transferred to the next generation, the nucleic acids have to undergo processes such as replication and recombination. All living systems contain a set of enzymes called nucleases, capable of interacting with nucleic acids and hydrolyzing the phosphodiester linkages. The enzymatic breakdown of nucleic acids was first observed in early twentieth century (Araki, 1903) and the term "nucleases" was coined for enzymes involved in the degradation of nucleic acids. However, it was not until 1940, that Kunitz (1940, 1950) described two groups of nucleases, based on sugar specificity. Different schemes of classification were proposed in an attempt to overcome the shortcomings of the earlier ones (Kunitz, 1950 and Laskowski, 1959, 1967). However, with the discovery of newer nucleases and multifunctional enzymes like micrococcal nuclease and snake venom phosphodiesterase, the classification of Kunitz was found to be inadequate. Soon, a new class of sugar non-specific nucleases had to be added to the list as per new evidences. Hence, in order to overcome these shortcomings, Bernard (1969) and Laskowski (1959, 1982) suggested that nucleases be classified on the basis of - (i) The nature of substrate hydrolyzed (DNA, RNA) (ii) The type of nucleolytic attack (exonuclease and endonuclease) (iii) The nature of the hydrolytic products formed i.e. mono or oligo nucleotides terminating in a 3'- or a 5'- phosphate and (iv) The nature of the bond hydrolyzed. However, these schemes of classification did not make any provision for the difference in double-stranded (ds) and single-stranded (ss) cleavage. The credit for the discovery of nucleases hydrolyzing single-stranded nucleic acids goes to Lehman (1960). With the realization of the varied and complex nature of the catalytic activities of different nucleases, it became obvious that 6 exceptions did exist in almost every category of each of the proposed classification schemes which lead Laskowski (1982) to comment that the issue of classification expired because "the progress just overgrew all boundaries". Single-strand-specific nucleases are ubiquitous in distribution. They exhibit high selectivity for single-stranded nucleic acids and single-stranded regions in double-stranded nucleic acids (Shishido and Ando, 1985) and hence they are widely used as probes for the structural determination of nucleic acids, mapping mutations and studying the interactions of DNA with various intercalating agents (Drew, 1984). Intracellularly, some of them have been implicated in recombination, repair (West, 1985) and replication (Brown et. al., 1985). Although, their widespread use has lead to the isolation of more than 30 single-strand-specific nucleases from various sources, only a few enzymes such as S1 nuclease from Aspergillus oryzae, P1 nuclease from Penicillium citrinum, BAL 31 nuclease from Alteromonas espejiana, Neurospora crassa, Ustilago maydis and mung bean nucleases have been characterized sufficiently. Off late, a number of these enzymes have been cloned, their crystal structures solved and their interaction with different substrates has been well established. The present review gives a comprehensive account of single-strand-specific nucleases studied to date. Occurrence and localization It is well known that nucleases play an important role in the four R's i.e. recombination, replication,