The present thesis deals with different aspects of the chemistry of oxovanadium(IV) complexes, their interaction with DNA and protein and photo-induced DNA and protein cleavage activity and photocytotoxicity. Chapter I presents a general introduction on various modes of interactions of organic compounds and transition metal complexes capable of targeting DNA leading to DNA strand scission, emphasizing particularly the photo-induced DNA cleavage activities for their potential application in PDT. The mechanistic pathways associated with the DNA cleavage are discussed. A comparison has been made on the advantages of photoactive metal complexes over organic conjugates. Objective of the present investigation is also dealt in this Chapter. Chapter II of the thesis deals with the synthesis, characterization, DNA binding and photo-induced DNA cleavage activity of ternary oxovanadium(IV) complexes of N-salicylidene-S-methyldithiocarbazate (salmdtc) and phenanthroline bases to explore the photo-induced DNA cleavage activity in UV-A light of 365 nm. Chapter III presents the synthesis, characterization, DNA binding and photo-induced DNA cleavage activity of ternary oxovanadium(IV) complexes containing N-salicylidene-L-methionate (salmet) and N-salicylidene-L-tryptophanate (saltrp) Schiff bases and phenanthroline bases. The objective of this work is to investigate the photo-induced DNA cleavage activity in near-IR light and to see the effect of pendant thiomethyl and indole moieties in the DNA cleavage reactions. Chapter IV deals with the synthesis, characterization, DNA binding, red-light induced DNA cleavage activity and photocytotoxicity of ternary oxovanadium(IV) complexes having N-salicylidene-L-arginine (sal-argH) and N-salicylidene-L-lysine (sal-lysH) Schiff bases and phenanthroline bases. The important results include the visible light-induced DNA cleavage activity and photocytotoxicity of the complexes in human cervical HeLa cancer cells. Chapter V describes the synthesis, characterization, DNA binding and photo-induced DNA and protein cleavage activity and photocytotoxicity of oxovanadium(IV) complexes containing bis(2-benzimidazolylmethyl)amine and phenanthroline bases. The significant results include DNA cleavage activity in near-IR light and photocytotoxicity of the dppz complex in non-small cell lung carcinoma/human lung adenocarcinoma A549 cells in visible light. Further, we have studied the protein cleavage activity of the complexes in UV-A light of 365 nm by using bovine serum albumin (BSA) and lysozyme. Finally, Chapter VI presents the binary oxovanadium(IV) complexes of phenanthroline bases. We have studied their synthesis, characterization, DNA binding and photo-induced DNA and protein cleavage activity and photocytotoxicity. Photocytotoxicity of dppz complex has been studied in human cervical HeLa cancer cells in visible light. Photo-induced protein cleavage activity of the complexes has been studied in UV-A light of 365 nm by using BSA and lysozyme. The references have been compiled at the end of each chapter and indicated as superscript numbers in the text. The complexes presented in this thesis are represented by bold-faced numbers. Crystallographic data of the complexes, characterized structurally by single crystal X-ray crystallography, are given in CIF format in the enclosed CD (Appendix-I). Due acknowledgements have been made wherever the work described is based on the findings of other investigators. Any omission that might have happened due to oversight or mistake is regretted.

The present thesis deals with different aspects of the chemistry of iron complexes, their interaction with DNA and photo-induced cleavage of double-stranded DNA. Chapter I presents a general introduction on metal-based drugs in cancer therapy and the evolution of the transition metal complexes capable of targeting DNA leading to DNA strand scission, emphasizing particularly the photo-induced DNA cleavage activities for their potential utility in PDT. The mechanistic pathways associated with the DNA cleavage are discussed citing selected examples of compounds that are known to be efficient DNA photo-cleavers on irradiation with light of different wavelengths. Objective of the present investigation is dealt in this chapter. Chapter II deals with the synthesis, crystal structure, DNA binding and oxidative DNA cleavage activity of ternary iron(II) complexes of phenanthroline bases to explore the chemistry of iron(II) complexes towards the metal-assisted photo-induced DNA cleavage activity. Chapter III presents the synthesis and characterization of a cationic imidazo[1,5-a]pyridine derivative to explore the role of imidazopyridine moiety in the photo-induced DNA cleavage activity of the compound. Its cytotoxic effect to the HeLa cancer cell has also been studied using UV-A light of 365 nm. Chapter IV presents the synthesis and characterization of dipyridoquinoxaline (dpq) complexes of bivalent 3d-metal ions such as d6-iron(II), d7-cobalt(II), d8-nickel(II), d9-copper(II) and d10-zinc(II) to explore any specific role that is played by the transition-metal ions in exhibiting visible light-induced DNA cleavage activity. Chapter V deals with the synthesis, characterization of oxo-bridged diiron(III) complexes of phenanthrolne bases having a structural motif found in many iron-containing metalloproteins with a diiron core in the active site. DNA binding and photo-induced DNA cleavage activity of the complexes is studied. Finally, Chapter VI deals with the synthesis and characterization of oxo-bridged diiron(III) complexes having L-histidine (L-his) and N,N-donor heterocyclic bases. This chapter describes the double-strand DNA cleavage activity of [{Fe(L-his)(dpq)}2(μ-O)](ClO4)2. Rationalization of the DNA double strand break (dsb) has been made using molecular docking calculations. This chapter also deals with the site-specific protein (bovine serum albumin, BSA) cleavage activity of the complexes on UV-A light irradiation. The references have been compiled at the end of each chapter and indicated as superscript numbers in the text. The complexes presented in this thesis are represented by bold-faced numbers. Crystallographic data of the complexes, characterized structurally by single crystal X-ray crystallography, are given in CIF format in the enclosed CD (Appendix-I). Due acknowledgements have been made wherever the work described is based on the findings of other investigators. Any omission that might have happened due to oversight or mistake is regretted.

Structural and mechanistic complexities of copper-dioxygen systems have attracted much attention in the field of bioinorganic chemistry, both in model systems and trapped protein intermediates. The research presented herein is focused on model and naturally occurring metallopeptide systems, from its design to catalysis. Copper is used as the coordinating metal ion, with cobalt and zinc as probes for metal binding. The bioinorganic chemistry of copper proteins and its coordination and spectroscopic properties are briefly discussed in chapter 1. The next two chapters are centered on the de novo design of a minimalistic metallopeptide system with an amino acid sequence of RHHPPHHE. Structural characterization of the peptide by means of CD and NMR spectroscopy techniques are presented in chapter 2, suggesting a characteristic beta-turn structure in its apo and di-metal bound form. The designed metallopeptide exhibits catecholase activity, which is presented in chapter 3. The data suggest the presence of two mononuclear copper active sites, exhibiting specificity towards the oxidation of catecholamine substrates. Similarly, the catecholase activity has been previously observed in copper complexes of Alzheimer's disease related peptide beta-amyloid, exhibiting metal-centered redox chemistry. The metallo-(beta-amyloid); complexes are the hallmark Alzheimer's disease and have been attributed to the generation of reactive oxygen species causing oxidative stress. Thus, inhibition of the observed oxidative activities was investigated. Probing the role of phosphate moieties in various compounds as potential inhibitors against the induced oxidative stress is presented in chapter 4. The phosphate analogs of the studied compounds exhibit more pronounced potency, where mutation of the beta amyloid peptide at Arg-5 and Lys-16 give insight into the interactions of the side chains of Arg and Lys with the phosphate moiety. 31P NMR relaxation studies further support the binding/interaction of phosphate with the Cu(II)-(beta-amyloid); complexes. The correlation of phosphate moiety binding/activity will allow for the design of more potent inhibitors toward the Cu(II)-(beta-amyloid); induced oxidative stress.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24833号 / 医博第5001号 / 新制||医||1067(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田, 宏一, 教授 上野, 英樹, 教授 濵﨑, 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Photo sensitizers showing visible light induced DNA cleavage activity are of current importance for medicinal applications related to photodynamic therapy (PTD) considering greater skin penetration of light near 700 nm. While organic molecules and complexes of 4d-5d metal ions are extensively studied for their DNA photo-damage properties in UV and visible light, the chemistry of 3D metal complexes showing visible light-induced DNA cleavage activity is relatively unexplored efforts have been made in this thesis work to design new ternary copper (II) complexes having a-amino acids Such copper (II) complexes with tunable coordination geometry could find potential applications in PDT. Ternary Copper (II) complexes containing L-methionine, S-methy1-L-cysteine and phenanthroline bases are prepared and characterized. They display DNA binding and visible light induced DNA cleavage activity. An enhancement of the DNA cleavage activity is observed for analogous ternary copper (II) complexes contained L-lysine with a pendant cationic amine moiety as a photo-induced DNA Cleavage activity using binary and ternary copper (II) complexes of L-arginine and phenanthroline bases. We have observed AT selective DNA binding and visible light –induced DNA cleavage activity. The crescent-shaped bis-arginine Copper (II) complex mimics the natural antiviral antibiotic netropsin. T o investigate the role of the pendant groups of the amino acids, we have explored the DNA binding and DNA cleavage activity of analogues L-glutamine and L-asparagine complexes. We have prepared ternary copper (II) complexes containing two photosensitizers, viz., L-tryptophan (L-trp) and dipyridoquininoxaline/dipyridophenazine to achieve double strand breaks forming linear DNA. Complex [Cu(L-trp)(dppz)(H2O)+ shows a stacking arrangement of the indole and dppz rings giving a separation that fits with the base pair separation of ds-DNA. Photosensitizes in these complexes approach two different complementary stands of the ds-DNA, leading to double strand breaks and formation of linear DNA. Mechanistic studies on the DNA photocleavage reactions reveal the formation of singlet oxygen(1O2)species by a type-II pathway in preference to the hydroxyl radical generation. A process leading to an efficient DNA cleavage activity on visible light irradiation. The observation of sequence selectivity and double strand DNA cleavage on red light exposure by national design of the complexes is significant considering importance of the results in the chemistry of photodynamic therapy of cancer. The results of this dissertation open up new avenues for designing and developing 3d metal-based photosensitizers with potential utility in nulcleic acid chemistry.

In bacteria, nucleoid associated proteins (NAPs) represent a prominent group of global regulators that perform the tasks of genome compaction, establishing chromosomal architecture and regulation of various DNA transactions like replication, transcription, recombination and repair. HU, a basic histone like protein, is one of the most important NAPs in Eubacteria. Mycobacterium tuberculosis produces a homodimeric HU (MtbHU), which interacts with DNA non-specifically through minor groove binding. Exploration for essential genes in Mtb (H37Rv) through transposon insertion has identified HU coding gene [Rv2986c, hupB; Gene Id: 15610123; Swiss-Prot ID: P95109)] to be vital for the survival and growth of this pathogen. MtbHU contains two domains, the N-terminal domain which is considerably conserved among the HU proteins of the prokaryotic world, and a C–terminal domain consisting of Lys-Ala rich multiple repeat degenerate motifs. Sequence analysis carried out by the thesis candidate showed that MtbHU exhibits 86 to 100 percent identity within the N-term region among all the mycobacterium species and some of the members of actinobacteria, including important pathogens like M. tuberculosis, M. leprae, M. ulcerans, M. bovis, Nocardia; while C term repeat region varies relatively more. This strikingly high cross species identity establishes the MtbHU N-terminal domain (MtbHUN) as an important representative structural model for the above mentioned group of pathogens. The thesis candidate has solved the X-ray crystal structure of MtbHUN, crystallized in two different forms, P2 and P21. The crystal structures in combination with computational analyses elucidate the structural details of MtbHU interaction with DNA. Moreover, the similar mode of self assembly of MtbHUN observed in two different crystal forms reveals that the same DNA binding interface of the protein can also be utilized to form higher order oligomers, that HU is known to form at higher concentrations. Though the bifunctional interface involved in both DNA binding and self assembly is not akin to a typical enzyme active site, the structural analysis identified key interacting residues involved in macromolecular interactions, allowing us to develop a rationale for inhibitor design. Further, the candidate has performed virtual screening against a vast library of compounds, and design of small molecules to target MtbHU and disrupt its binding to DNA. Various biochemical, mutational and biological studies were performed in the laboratory of our collaborator Prof. V. Nagaraja, MCBL, IISc., to investigate these aspects. After a series of iterations including design, synthesis and validation, we have identified novel candidate molecules, which bind to MtbHU, disrupt chromosomal architecture and arrest M. tuberculosis growth. Thus, the study suggests that, these molecules can serve as leads for a new class of DNA-interaction inhibitors and HU as a druggable target, more so because HU is essential to Mtb, but absent in human. Our study proposes that, targeting the nucleoid associated protein HU in Mtb can strategize design of new anti-mycobacterial therapeutics. Perturbation of MtbHU-DNA binding through the identified compounds provides the first instance of medium to small molecular inhibitors of NAP, and augurs well for the development of chemical probe(s) to perturb HU functions, and can be used as a fundamental chemical tool for the system level studies of HU-interactome. Section I: “Crystal structure of Mycobacterium tuberculosis histone like protein HU and structure based design of molecules to inhibit MtbHU-DNA interaction: Leads for a new target.” of this thesis presents an elaborate elucidation of the above mentioned work. The candidate has additionally carried out structure based computational and theoretical work to elucidate the interaction of amino acid based metal complexes which efficiently bind to DNA via minor-groove, major-groove or base intercalation interaction and display DNA cleavage activity on photo-irradiation. This understanding is crucial for the design of molecules towards Photodynamic Therapy (PDT). PDT is an emerging method of non-invasive treatment of cancer in which drugs like Photofrin show localized toxicity on photoactivation at the tumor cells leaving the healthy cells unaffected. The work carried out in our group in close collaboration with Prof. A.R. Chakravarty of Inorganic and Physical Chemistry Department elaborates the structure based design of Amino acid complexes containing single Cu (II), such as [Cu(L-trp)(dpq)(H2O)]+ , [Cu (L-arg) 2](NO3)2 , Amino acid complexes containing oxobridged diiron Fe(III), such as [{Fe(L-his)(bpy)}2(μ-O)](ClO4)2 , [{Fe(L-his)(phen)}2(μ-O)](ClO4)2 , and Complexes containing Binuclear Cu(II) coordinated organic moiety, such as [{(dpq) CuII}2(μ-dtdp)2], which bind to DNA through minor groove/major groove/base intercalation interactions. Docking analysis was performed with the X-ray crystallographic structure of DNA as receptor and the metal complexes as ligands, to study the mode of binding to DNA and to understand the possible mode of DNA cleavage (single/double strand) when activated with laser. Section II: “Structure based computational and theoretical analysis of metal coordinated complexes containing amino acids and organic moieties designed for photo induced DNA cleavage” of this thesis presents a detailed presentation of the above mentioned work.

Scorpionate and terpyridine ligands are of importance in inorganic chemistry for their metal-binding properties. Tris-pyrazolylborate (Scorpionate) ligands that show facial binding mode and steric protection have been extensively used to synthesize complexes modeling the active site structure and biological function of various metalloproteins and as catalysts in C-H and NO activation and carbine transfer reactions. Terpyridine and modified terpyridine ligands showing meridional binding mode have been used in bioinorganic chemistry where Pt-terpyridine complexes are known to inhibit the activity of thiordoxin reductase (TrxR) besides showing interaction with G-quadruplex. The thesis work stems from our interest to use these ligand systems to design and prepare new 3-d metal-based photodynamic therapeutic (PDT) agents to explore their visible light-induced DNA cleavage activity and photocytotoxicity. Efforts have been made in this thesis work to design and synthesize Co(II) and Cu(II) complexes having scorpionate (Tpph) abd terpyridine (tpy) ligands. Ternary 3d-metal complexes having Tpph and planar phenanthroline bases have been synthesized and structurally characterized. The steric encumbrance of Tpph has led to the reduction in chemical nuclease activity along with enhanced photo-induced DNA cleavage activity, particularly of the Cu(II) and Co(II) complexes. The Co(II), Cu(II) and Zn(II) complexes of Tpph and a pyridyl ligand having a photoactive naphthalilmide moiety show molecular light-switch effect on binding to calf thymus DNA or BSA protein. The complexes do not show any chemical nuclease activity. The Cu(II) complex shows significant DNA cleavage activity in red light. The Co(II) complex displays significant photocytotoxicity in UV-A light. Ternary Cu(II) complexes of ph-tpy and heterocycylic bases are prepared and their DNA binding and cleavage activity studied. The complexes are avid binders to CT-DNA. The dipyridoquinoxaline (dpq) and dipyridophenazine (dppz) complexes are photocleavers of DNA in visible light. A significant enhancement in cytotoxicity in HeLa cancer cells is observed on exposure of the dppz complex to light. The binary Cu(II) complexes are also prepared to reduce the dark toxicity using phenyl and pyrenyl substituted terpyridine ligands. The pyrenyl substituted complex shows DNA cleavage activity in the visible light, low dark toxicity and unprecedented photocytotoxicity in visible light. The copper(II) complexes generally show dark cellular toxicity due to the presence of reducing thiols. The present terpyridine copper(II) complex having pendant pyrenyl moiety shows significant PDT effect that is similar to that of the PDT drug Photofrin. Binary Co(II) complexes show efficient DNA cleavage activity in visible light, significant photocytotoxicity in visible light and cytosolic uptake behaviour. Considering the bio-essential nature of the cobalt and copper ions, the present study opens up new strategies for designing and developing 3d-metal-based photosensitizers for their potential applications in PDT.

In this work, six triazene compounds are assayed to DNA cleavage activity. The same six triazenes and the drug Asercit® (dacarbazine) are assayed to antibacterial activity and toxicity to Artemia salina Leach.: 1,3-bis-(phenyl)triazene-1-N-hidroxide (T1), 1-(4-bromophenyl)-3-(4-nitrophenyl)triazene, (T2), 1-(4-azophenyl)-3-(4-nitrophenyl)triazene (T3) ,1,3-bis-(4-azophenyl-triazene) (T4), 1,3-bis-(2-bromophenyl)triazene (T5), 1-(4-carboxyphenyl)-3-(4-azophenyl)triazene (T6) and 5-(3,3-dimethyl-1-triazenyl)imidazol-4-carboxamide (dacarbazine, Asercit®). Triazene T1 cleaves approximately 50 % of plasmid DNA (pBSKII and pUC18, 3.75 mM, 50ºC/24 hours, Tris.HCl buffer 200 mM pH 8.0). Hydroxyl radical scavengers (glycerol 0.1 and 1.0 %, DMSO 0.04 M and tiourea 0.04 M) and argon atmosphere not interfere in DNA cleavage by T1. The Kb of T1 determinated by spectrofotometric titrations with DNA are 4.50 x 101 M-1 (pH 6,5), 1.00 x 102 M-1 (pH 7,0) e 2.33 x 102 M-1 (pH 8,0). T1, T6 and Asercit® are the more actives in antibacterial activity with CIM/CBM of even 16/64 μg/ml. LC50 more lower determinated by toxicity to A. salina are to T1 (0.081 ± 0.008 μg/ml), T5 (0.076 ± 0.011 μg/ml) and T6 (0.077 ± 0.007 μg/ml). Theses studies show the wide biological activity conferred by triazene compounds. / Neste estudo, seis compostos triazenos são avaliados quanto à atividade de clivagem do DNA. Os mesmos seis triazenos e o medicamento Asercit® (dacarbazina) são avaliados quanto à atividade antibacteriana e toxicidade frente à Artemia salina Leach.: 1,3-bis-(fenil)triazeno-1-N-hidróxido (T1), 1-(4-bromofenil)-3- (4-nitrofenil)triazeno (T2), 1-(4-azofenil)-3-(4-nitrofenil)triazeno (T3), 1,3-bis-(4- azofenil)triazeno (T4), 1,3-bis-(2-bromofenil)triazeno (T5), 1-(4-carbóxifenil)-3-(4- azofenil)triazeno (T6) e 5-(3,3-dimetil-1-triazenil)imidazol-4-carboxamida (dacarbazina, Asercit®). O triazeno T1 cliva aproximadamente 50 % do DNA plasmidial (pBSKII e pUC18, a 3,75 mM, 50 ºC/24 horas, tampão Tris.HCl 200 mM pH 8,0). Seqüestradores de radicais hidroxil (glicerol 0,1 e 1 %, DMSO 0,04 M e tiouréia 0,04 M) e atmosfera de argônio não interferem na clivagem do DNA por T1. Os Kb de T1 determinados por titulação espectrofotométrica com DNA são 4,50 x 101 M-1 (pH 6,5), 1,00 x 102 M-1 (pH 7,0) e 2,33 x 102 M-1 (pH 8,0). T1, T6 e o Asercit® são os mais ativos na atividade antibacteriana com CIM/CBM de até 16/64 μg/ml. As LC50 mais baixas determinadas pela toxicidade para A. salina são para T1 (0,081 ± 0,008 μg/ml), T5 (0,076 ± 0,011 μg/ml) e T6 (0,077 ± 0,007 μg/ml). Esses estudos mostram a ampla atividade biológica conferida pelos compostos triazenos.

No description available.

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.