Restriction modification (R-M) systems are important components of the prokaryotic arsenal against invading genomes. R-M systems directly target the foreign DNA and are often considered as primitive immune systems in bacteria. The defense system comprises of two contrasting enzymatic activities – a restriction endonuclease (REase) and a methyltransferase (MTase). Functionally, REases cleave a specific DNA sequence endonucleolytically at the phosphodiester bonds generating 5' or 3' overhangs or blunt ends. MTases catalyze the transfer of a methyl group from S-adenosyl-Lmethionine to adenine or cytosine. Four types of R–M systems are found in bacteria, viz., Types I, II, III and IV. Type II R-M systems, comprising of a separate REase and MTase, are the most abundant and well-studied enzymes. Type II REases recognize and cleave DNA within or near their recognition sequences. Surprisingly, these enzymes share little or no sequence homology amongst them. All the enzymes identified so far can be grouped into conventional PD-(D/E)XK, ββα-Me, GIY-YIG, phospholipase-derived and half-pipe endonucleases according to their folds and active site structures. Owing to their high specificity and defined cleavage pattern, they have become indispensable tools in molecular biology and have been widely exploited for studying protein–DNA interactions. The work presented in this thesis deals with R.KpnI, which belongs to the HNH superfamily of nucleases and is characterized by the presence of a ββα-Me finger motif. The REase isolated from Klebsiella pneumoniae recognizes the palindromic DNA sequence GGTAC/C and cleaves DNA as indicated. The enzyme is unique in exhibiting promiscuous DNA cleavage in the presence of Mg2+, a natural co-factor for a vast majority of REases. Surprisingly, Ca2+ and Zn2+ completely suppress the Mg2+ mediated promiscuous activity and induce high fidelity cleavage. These unusual features of R.KpnI led to the functional characterization of the ββα-Me finger active site motif. In addition, the studies were aimed at understanding the mechanism and the biological significance of substrate and co-factor promiscuity exhibited by the enzyme. The salient aspects of the thesis are summarized below. A general introduction and overview of the literature on structure-function studies, mechanism of recognition and catalysis by REases with special emphasis on Type II enzymes is presented in the Chapter 1. An account of co-factor specificity in REases, role of metal ions in DNA binding as well as in phosphodiester bond hydrolysis is provided. The various aspects of R-M systems that target the invading DNA elements and counter strategies employed by the foreign genomes to evade the restriction are also covered. The new developments that provide insights in understanding the diversity of R-M systems and additional biological roles that could increase the fitness of the host organism harboring them are described. The features of substrate and metal ion specificity in REases and the efforts undertaken to alter the specificity have been dealt at the end of the chapter. From the structures of the several ββα-Me finger nucleases, the α-helix has been implicated in providing a structural scaffold for the correct juxtapositioning of the catalytic residues. However, no mutagenesis data exists to delineate its role. Homology modeling studies of R.KpnI suggested a crossover structure for the α-helix of the ββαMe finger active site motif, which could possibly form dimeric interface and/or structural scaffold for the active site. Chapter 2 describes the computational modeling and mutational analysis performed to understand the role of the residues present in this α-helix in intersubunit interactions and/or stabilization of the active site. Mutation of the residues present in the α-helix lead to the loss of the enzyme activity, but not dimerization ability. Subsequent biophysical experiments showed that the α-helix of the ββα-Me finger of R.KpnI plays an important role for the stability of the protein–DNA complex needed for its function. In Chapter 3, unusual co-factor flexibility for R.KpnI is shown by using a battery of divalent metal co-factors differing in ionic radii and coordination geometries. A number of alkaline earth and transition group metal ions function as co-factors for DNA cleavage. The metal ions replaced each other readily from the enzyme’s active site revealing the active site plasticity. Mutation of the invariant His residue of the HNH motif caused abolition of the enzyme activity with all the co-factors indicating that the enzyme follows single metal ion mechanism for DNA cleavage. The indispensability of the invariant His in nucleophile activation together with the broad co-factor tolerance of the enzyme indicated the role of metal ions in electrostatic stabilization during catalysis. At higher concentrations, Mg2+, Mn2+ or Co2+ stimulate promiscuous cleavage while Cd2+, Ni2+ or Zn2+ inhibit phosphodiester bond hydrolysis. The underlying molecular mechanisms for the modulation of the enzyme activity by the metal ion binding to the second site are presented. Regulation of the endonuclease activity and fidelity by a second metal ion binding is a unique feature of R.KpnI among REases and HNH nucleases. The identification of additional metal ion binding residues would help in engineering REase variants with enhanced activity and/or specificity. Chapter 4 describes the generation of an R.KpnI variant with altered co-factor specificity by exploiting the active site plasticity of the enzyme. The mutant enzyme is a Mn2+ -dependent endonuclease defective in DNA cleavage with Mg2+ and other divalent metal ions. In the engineered mutant, only Mn2+ is selectively bound at the active site, imparting in vitro activity while being dormant in vivo. In addition to the Mn2+ selectivity, the mutant is impaired in concerted double-stranded DNA cleavage leading to the accumulation of nicked intermediates. The nicking activity of the mutant enzyme is further enhanced by altering the reaction conditions. Thus, a single point mutation in the active site of R.KpnI generates a Mn2+ -dependent REase and a sequence specific nicking endonuclease. The potential applications of such enzymes engineered for selective metal ion dependent activities have been discussed. R.KpnI is peculiar in retaining robust promiscuous cleavage despite being a typical Type II REase in all other characteristics. Chapter 5 presents results of the growth properties and phage titer analysis carried out with R.KpnI and its high fidelity variant to understand the biological significance of promiscuous activity. The enzyme isolated from the K. pneumoniae exhibited biochemical properties similar to that of R.KpnI overexpressed in E.coli. It was observed that the wild type but not the high fidelity variant could effectively restrict bacteriophages methylated at GGTACC. These results show that the REase exhibits promiscuous activity in vivo, which would be advantageous for the organism to better target the incoming foreign DNA. The promiscuous behavior of the R.KpnI could be one of the counter strategies employed by the bacteria against the constantly evolving phages in the co-evolutionary arms race. In conclusion, the work described in this thesis provides new insights about structure, function and biology of REases in general and R.KpnI in particular. The co-factor and substrate promiscuity of R.KpnI may indicate its evolutionarily intermediate form that is yet to attain a high degree of specificity. Alternatively, it is possible that this unique feature is retained during the evolution of the HNH REases serving some unknown function(s) in the cell, in addition to having an edge in countering the phage infections.

Bacteria employ survival strategies to protect themselves against foreign invaders, including bacteriophages. The ‘immune system’ of bacteria relies mostly on restriction-modification (R-M) systems. The primary role of R-M systems is to protect the host from invading foreign DNA molecules. Three major types of R–M system are found in bacteria, viz.Types I, II and III. Type II R–M systems comprise a separate restriction endonuclease (REase) and a methyltransferase (MTase) that act independently of each other. Type II REases generally recognize palindromic sequences in DNA and cleave within or near their recognition sequences and produce DNA fragments of defined sizes. They have become indispensable tools in molecular biology and have been widely exploited for studying site-specific protein–DNA interactions. Surprisingly, these enzymes share little or no sequence homology among them, though the three-dimensional structures determined to date reveal a common-core motif (‘PD...D/EXK’ motif) with a central β-sheet that is flanked by α-helices on both sides. In the motif, two acidic residues (D and D/E) are important for the metal ion binding and catalysis. The work presented in this thesis deals with the determination of active site, elucidation of kinetic mechanism and study of evolution of sequence specificity using the well known, R.KpnI, from Klebsiella pneumoniae. The enzyme is a homodimer, which recognizes a palindromic double stranded DNA sequence, GGTAC↓C, and cleaves as shown. Unlike other REases, R.KpnI shows prolific promiscuous DNA cleavage in presence of Mg2+. Surprisingly, Ca2+ completely suppresses the Mg2+ mediated promiscuous activity and induces high fidelity cleavage at the recognition sequence. These unusual properties of R.KpnI led to the characterization of the active site of the enzyme. This thesis is divided into five chapters. Chapter 1 is a general introduction of R-M systems and an overview of the literature on active sites of Type II REases. It deals with discovery, nomenclature and classification followed by description of the enzymes diversity and general features of Type II REases. The different active site folds of the REases have been discussed in detail. The features of sequence specificity and the efforts undertaken to engineer the new specificity in the REases have been dealt at the end of the chapter. Chapter 2 describes identification and characterization of the R.KpnI active site by bioinformatics analyses, homology modeling and mutational studies. Bioinformatics analyses reveal that R.KpnI contains a ββα-Me-finger fold, which is a characteristic of many HNH-superfamily endonucleases. According to the homology model of R.KpnI, the putative active site residues correspond to the conserved residues present in HNH nucleases. Substitutions of these conserved residues in R.KpnI resulted in loss of the DNA cleavage activity, confirming their importance. This study provides the first experimental evidence for a Type IIP REase that is a member of the HNH superfamily and does not belong to the PD...D/EXK superfamily of nucleases. In Chapter 3 DNA binding and kinetic analysis of R.KpnI is presented. The metal ions which exhibit disparate pattern of DNA cleavage have no role in DNA recognition. The enzyme binds to both canonical and non-canonical DNA with comparable affinity irrespective of the metal ions used. Further, it was shown that Ca2+-imparted exquisite specificity of the enzyme is at the level of DNA cleavage and not at the binding step. The kinetic constants were obtained through steady-state kinetic analysis of R.KpnI in presence of different metal ions. With the canonical oligonucleotides, the cleavage rate of the enzyme was comparable for both Mg2+- and Mn2+-mediated reactions and was about three times slower with Ca2+. The enzyme discriminates non-canonical sequences poorly from the canonical sequence in Mg2+-mediated reactions unlike any other Type II REases, accounting for its promiscuous behavior. These studies suggest that R.KpnI displays properties akin to that of typical Type II REases and also endonucleases with degenerate specificity for DNA recognition and cleavage. In chapter 4, two uncommon roles for Zn2+ in R.KpnI are described. Examination of the sequence revealed the presence of a zinc finger (CCCH) motif rarely found in proteins of prokaryotic origin. Biophysical experiments and subsequent mutational analysis showed that the zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the Mg2+- and Mn2+-mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in a REase. Chapter 5 describes generation of highly sequence specific R.KpnI. Towards this end, site-directed mutants were generated at the putative secondary metal binding site. The DNA binding and cleavage analyses of the mutants at putative secondary metal binding site revealed that the secondary site is not important for primary catalysis and have a role in sequence specificity. A single amino acid change at the D163 position abolished the promiscuous activity of the wt enzyme in the presence of Mg2+ and Mn2+. Thus, a single point mutation converts the promiscuous endonuclease to a high fidelity REase. In conclusion, the work described in the thesis reveals new information on the REases in general and R.KpnI in particular. Many of the properties of R.KpnI elucidated in this thesis represent hitherto unknown features amongst REases. The presence of an HNH catalytic motif in the enzyme indicates the diversity of active site fold in REases and their distinct origin. Similarly, the high degree of promiscuity exhibited by the enzyme may hint at the evolutionary link between non-specific and highly sequence specific nucleases. The present studies also provide an example for the role of mutations in the evolution of sequence specificity. The utilization of different metal ions for DNA cleavage and the architectural role for Zn2+ in maintaining the structural integrity are other unusual properties of the enzyme.