The fidelity of DNA replication prevents mutations that may lead to cancer predisposition or neurodegenerative diseases. One mechanism that enhances DNA replication fidelity is DNA mismatch repair, which corrects mismatches and small insertion/deletion loops that have escaped polymerase proofreading. In all eukaryotes and most prokaryotes, MutL (a key mismatch repair protein) has an intrinsic endonuclease activity that nicks the newly synthesized strand and recruits downstream factors to remove and correct errors. It has been proposed that ATP binding promotes a series of conformational changes that induce structural order within MutL and stimulates its endonuclease activity. The C-terminal domain of MutL, which harbors the endonuclease site, does not bind to DNA. This has prevented the molecular characterization of its endonuclease activity. In this thesis, we first show that MutL in B. subtilis exhibits asymmetric conformations similar to yeast and human MutL homologs. We also devise a novel approach to bypass the binding defect of the C-terminal domain by using fusion proteins. We find that these fusions bind to DNA specifically and, in the presence of the processivity clamp, can nick DNA. One of these fusion proteins in particular stimulates the nicking activity much more efficiently than the C-terminal domain alone. This work lays the foundation for the mechanistic characterization of the MutL endonuclease and provides a method to stabilize transient protein-DNA interactions. / Thesis / Master of Science (MSc)

Restriction endonucleases occur ubiquitously among bacteria, archaea and in viruses of certain unicellular algae, and they are usually accompanied by a modification enzyme of identical specificity; together, the two activities form a restriction-modification (R-M) system- the prokaryotic equivalent of an immune system. More than 3,800 R-M enzymes have been characterized so far and they manifest 262 unique recognition specificities. These enzymes represent the largest family of functionally related enzymes. Based on the number and organization of subunits, cofactor requirements, catalytic mechanism, and sequence specificity, restriction enzymes have been classified into different types, Types I, II, III, and IV. R-M systems are important model systems for studying highly specific DNA-Protein interactions and serve as excellent systems for investigating structure-function relationship and for understanding the evolution of functionally similar enzymes with highly dissimilar sequence. In bacteria, DNA methyltransferases (MTases) associated with R-M systems protects the host DNA from cleavage by the cognate restriction endonuclease recognizing the same sequence and provides the integrity of host cell genome against foreign DNA invasion. The modification MTases catalyses the addition of a methyl group to one nucleotide in each strand of the recognition sequence using S-adenosyl-L-methionine (AdoMet) as the methyl group donor. Based on the chemistry of the methylation reaction catalyzed, DNA MTases are classified as C5 enzymes (endocyclic MTases), which transfer the methyl group to C5 position of cytosine, and N6 and N4 enzymes (exocyclic amino MTases), which transfer the methyl group to the exocyclic amino group of adenine or cytosine, respectively. DNA MTases of all three types contain conserved regions, which are responsible for catalysis and AdoMet binding, and variable regions known as target recognition domains (TRD), which determine the substrate specificity of a particular enzyme. Ten conserved amino acid motifs (I–X) are found in C5 MTases. Exocyclic DNA MTases are subdivided further into six groups (namely α, β, γ, ζ, δ and ε), according to the linear arrangements of three conserved motifs, the AdoMet-binding domain (FXGXG), the TRD (target recognition domain) and the catalytic domain (D/N/S)PP(Y/F). Base flipping has been proposed as a general mechanism used by all MTases in which the target base to be methylated is rotated 180º out of the DNA into a catalytic domain (motif IV). EcoP15I restriction enzyme (R.EcoP15I) belongs to the Type III restriction-modification (R-M) family. These enzymes are composed of two subunits, Res (Restriction) and Mod (Modification). The Mod subunit alone functions as a DNA methyltransferase in presence of AdoMet and magnesium and determines the specificity for restriction and methylation, whereas restriction activity requires the cooperation of both the Res and Mod subunits. EcoP15I methyltransferase (M.EcoP15I), a homodimeric enzyme catalyzes the transfer of a methyl group from AdoMet to the second adenine residue in the recognition sequence, 5’-CAGCAG-3’, in presence of magnesium ions. M.EcoP15I belongs to the β-subfamily of N6-adenine methyltransferases. In addition to the two highly conserved sequence motifs, FXGXG (motif 1) involved in AdoMet binding and DPPY (motif IV) involved in catalysis, the amino acid residues of the region 355-377 contains a PD(X)n(D/E)XK-like motif involved in metal binding. A Mutation in the Mod Subunit of EcoP15I Restriction Enzyme Converts the DNA Methyltransferase to a Site-Specific Endonuclease An interesting aspect of M.EcoP15I is that the methylation requires magnesium and magnesium binding to the PD(X)n(D/E)XK-like motif participates in base flipping. The PD-(D/E)XK superfamily of Mg2+-dependent nucleases were initially identified in structurally characterized Type II REases and later found in many enzymes involved in DNA replication, recombination and repair. The charged residues from the catalytic triads are implicated in metal ion mediated DNA cleavage. In EcoP15I DNA methyltransferase, a PD(X)n(D/E)XK like motif is present in which the partially conserved proline is replaced by methionine (MD(X)18(D/E)XK). Using site-directed mutagenesis methionine at 357 was changed to proline (M357P), which resulted in the formation of a Mg2+ binding/catalytic motif similar to several Mg2+-dependent endonucleases. Substitution of methionine at position 357 by proline converts EcoP15I DNA methyltransferase to a site-specific endonuclease. The mutant protein specifically binds to the recognition sequence 5’-CAGCAG-3’ and cleaves DNA in presence of Mg2+. The engineered EcoP15I-M357P is an active, sequence-dependent restriction endonuclease that cleaves DNA 10/1 nucleotide away from its recognition sequence in the presence of Mg2+. Unlike the holoenzyme, R.EcoP15I, the engineered endonuclease neither requires AdoMet or ATP nor requires two sites in the inverted orientation for DNA cleavage. It is of potential interest to use such an engineered enzyme as a genetic manipulation tool. Dimerisation of EcoP15I DNA Methyltransferase is Required for Sequence Recognition and Catalysis In the cell, after each round of replication, substrate for any DNA MTase is hemimethylated DNA and therefore, only a single methylation event restores the fully methylated state. This is in agreement with the fact that most of the DNA MTases studied exist as monomers in solution. The peculiar feature of M.EcoP15I is that it methylates only one strand of the DNA, at the N6-position of the adenine residue. Earlier studies using gel filtration and glutaraldehyde cross-linking demonstrated that M.EcoP15I exists as dimer in solution. However, the significance of dimerisation in the reaction mechanism of EcoP15I MTase is not clear. Therefore, experiments have been performed to determine whether M.EcoP15I could function as a monomer and the significance of dimerisation, if any, in catalysis. Towards this a homology model of the M.EcoP15I was generated by “FRankenstein monster” approach. Residues D223, V225, and V392, the side chains of which are present in the putative dimerisation interface in the model were targeted for site-directed mutagenesis. These residues were mutated to lysine and their importance was studied. Methylation and in vitro restriction assays showed that the triple mutant was catalytically inactive. Interestingly, the mutations resulted in weakening of the interaction between the monomers leading to both monomeric and dimeric species. M.EcoP15I was inactive in the monomeric form and therefore, dimerisation might be the initial step in its function. This must be required for positioning of the target base of the DNA in the active-site pocket of the M.EcoP15I. A part of this interface may be involved in site-specific DNA binding. Dimerisation of M.EcoP15I is, therefore, a prerequisite for the high-affinity substrate binding needed for efficient catalysis. Understanding the role(s) of Amino and Carboxyl-terminal Domains of EcoP15I DNA Methyltransferase in DNA Recognition and Catalysis N-terminal and C- terminal domains (NTD and CTD) of proteins are known to play many important roles such as folding, stability, dimerisation, regulation of gene expression, enzyme activity and substrate binding. From the modeled dimeric structure of M.EcoP15I, it was hypothesized that N- and C-termini are in close proximity with each other. In addition, it was predicted that each monomer can bind to AdoMet and DNA. Towards understanding the role(s) of the N- and C-terminal domains of M.EcoP15I in its structure and function, N-, and C-terminal deletions were created. Interestingly, deletion of N-terminal 53 amino acids and C-terminal 127 amino acids from of EcoP15I MTase converted the dimeric enzyme to a stable, monomeric protein that was structurally stable but enzymatically inactive. Each monomer could bind single-stranded DNA but dimerisation was required for double-stranded DNA binding and methylation. This indicated that amino acids at the N- and C-termini are important for maintaining a proper dimeric structure for M.EcoP15I functions. Therefore, it can be proposed that in a complex three-dimensional structure, the NTD and CTD should be properly maintained in order to execute its function, including dimerisation and DNA binding. However, since the 3D structure of M.EcoP15I has not yet been determined, the biochemical, biophysical and bioinformatics approaches may serve to provide useful information on the relative contributions of the electrostatic forces and hydrophobic contacts to the structural stability. Understanding the structural organization and folding of M.EcoP15I is crucial to elucidation of the mechanism of action.