In many DNA processes, action at a distance is required for signaling across long distances on DNA. These pathways, generally have an initiation site (site 1) that signals an event at a second location (site 2). Such a paradigm is found in processes such as transcription, replication, and DNA repair. To overcome long distances on DNA, proteins can utilize translocation, oligomerization, and DNA looping to bridge the distance between the initiating signal at site 1 and the site of action at site 2. The utilization of these mechanisms for action at a distance is crucial in eukaryotic mismatch repair. In this pathway, MutS homologs scan DNA and recognize mis-paired bases. The MutS protein then recruits the endonuclease MutLα, which nicks the nascent strand of DNA containing a mis-incorporated DNA base. The MutLα-generated nick leads to downstream mis-pair removal through excision by an exonuclease or strand displacement activities of a DNA polymerase working together with a flap endonuclease. Although, previous models have suggested that MutL homolog endonucleases can form oligomeric complexes on DNA, the role of a MutLα oligomeric complex and how it might facilitate action at a distance has been unclear. Here, I present evidence that the mismatch repair MutLα endonuclease is activated by DNA-DNA associations, and it can use this activity to overcome DNA torsional barriers. Using DNA ligation and pull-down experiments, I determined that a MutLα oligomer associates two DNA duplexes and that this activity can stimulate MutLα’s endonuclease function. I also show evidence that MutLα enhances a topoisomerase without nicking the DNA itself. These behaviors of MutLα could localize nicking on DNA near a mismatch and help overcome barriers that could inhibit additional repair proteins from activating MutLα and facilitating efficient DNA repair. The endonuclease activity of MutLα is critical for efficient mismatch repair, but in addition to this activity, MutLα is also an ATPase, although the crosstalk between the two enzymatic functions has been largely unexplored. It has been shown previously that the ATPase activity of MutLα allows the protein to undergo conformational changes and in vivo is necessary for efficient mismatch repair. Mechanistically, how this activity supports MutLα’s functions in the mismatch repair pathway remains unclear. Using DNA binding and photo-crosslinking experiments, I provide evidence that MutLα recognizes and localizes itself to a nick. Additionally, through DNA protection assays and photo-crosslinking I provide evidence of a signaling mechanism initiated at the nick for a MutLα oligomer to undergo its ATP cycle. These data provide insight into how MutLα uses ATP to signal events for mismatch removal. These data also provide a mechanistic explanation for how MutL proteins interact with DNA during mismatch repair and send signals for additional repair processes after the protein nicks DNA that help explain new models for action at a distance. / Chemistry

DNA encodes the genetic information of the cell, therefore, every single living organism has a precise DNA damage response mechanism to safeguard DNA integrity. Base mismatches are endogenous DNA lesions introduced by the replicative polymerase during DNA replication. The conserved DNA mismatch repair pathway corrects these base mismatches. Mismatch repair initiation is orchestrated by two proteins, MutS and MutL. MutS recognizes and binds to base mismatches and relays the presence of the lesion to MutL. MutL, in turn, interacts with downstream factors to coordinate mismatch excision. The processivity clamp, typically known for its role in tethering the DNA polymerase to DNA during replication, is also involved in several steps of this repair process including MutL endonuclease activation and strand resynthesis. The dynamics of the MutS-MutL and MutL-processivity clamp interactions present one of the bottlenecks to uncovering the spatial and time organization of these protein assemblies. Therefore, little is known about the interactions that orchestrate the early steps of mismatch repair. The biochemical and structural work included in this thesis outlines a precise series of molecular cues that activate MutL. / Thesis / Doctor of Philosophy (PhD)

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)

DNA mismatch repair corrects base-base mismatches and small insertion/deletion loops generated during normal DNA replication. If left unrepaired, these errors become permanent mutations and can lead to increased susceptibility to cancer. In most prokaryotes and all eukaryotes, the mismatch repair protein MutL is a sequence-unspecific endonuclease that plays an essential role in the strand discrimination step of this pathway. Prokaryotic MutL forms homodimers with two endonuclease sites, whereas eukaryotic MutL homologs form heterodimers with a single active site. To elucidate the mechanistic differences between prokaryotic and eukaryotic MutL, we tested whether both endonuclease sites are necessary for prokaryotic MutL nicking activity. MutL interaction with the processivity clamp is required to stimulate endonuclease activity. Therefore, we also tested whether both subunits of the MutL dimer needed to interact with the processivity clamp. To this end, we engineered a system to independently manipulate each protomer of the homodimer. We demonstrated that prokaryotic MutL is regulated by the processivity clamp to act in a similar manner to eukaryotic MutL with only one functional site contributing to the endonuclease activity. We also devised a strategy to stabilize the transient interactions between MutL, the β-clamp, and DNA through disulfide bridge crosslinking and heterobifunctional crosslinking. Stabilizing transient protein-protein and protein-DNA interactions will help optimize future structural studies in obtaining the ternary complex for mechanistic insights to the MutL endonuclease activity and regulation imposed by the β-clamp. / Thesis / Master of Science (MSc)

No description available.

DNA mismatch repair ensures faithful transmission of genetic material from parents to progeny, which is required for the survival of the organism. The studies on E. coli MMR proteins have formed the basis for the study of the MMR system in eukaryotic organisms, because the functions of MMR proteins believed to be been conserved. In organisms that harbor MutH protein, it is known that MutH acts as a monomer which nicks the unmethylated daughter strand and is activated in a MutS-MutL- dependent manner. The cleavage specificity of MutH is very stringent. Till recently, it was not clear as to how MutH distinguishes hemimethylated DNA from fully or unmethylated DNA. The co-crystal structures of MutH-DNA complexes revealed that Y212, R184 and P185 were in close proximity to the methyl-adenine. Clustal-W sequence alignment of MutH with Sau3AI showed that Sau3AI has PCT residues instead of L183, R184, and P185. A triple mutant MutH-L183P-R184C-P185T was found to cleave both unmethylated and methylated DNA. The nicking endonuclease activity of the LRP→ PCT triple mutant was enhanced in the presence of Haemophilus influenzae MutL. The mutL gene of Neisseria gonorrhoeae was cloned and the gene product purified. It was shown that the homodimeric Neisseria gonorrhoeae MutL (NgoL) protein displays an endonuclease activity that incises covalently closed circular DNA in the presence of manganese or magnesium or calcium ions unlike human MutLα which shows endonuclease activity only in the presence of manganese. Further more the C-terminal domain of Neisseria gonorrhoeae MutL (NgoL-CTD) consisting of amino acids 460 to 658 also exhibits Mn2+ dependent endonuclease activity. Sedimentation velocity, sedimentation equilibrium and dynamic light scattering experiments show NgoL-CTD to be a dimer. By in vitro comparison of wild-type and a mutant NgoL-CTD protein, it was shown that the latter protein exhibits highly reduced endonuclease activity. Surface plasmon resonance spectroscopy was used to determine the kinetics of DNA binding by NgoL. The DNA binding was carried out in absence of metal ions. Interaction studies with NgoL with ssDNA in SPR spectroscopy revealed a KD value of 4.7 × 10–8 M. While the human MutLα endonuclease activity was shown to be stimulated by ATP, ATP inhibits NgoL endonuclease activity. By in vitro comparison of wild-type and a mutant NgoL-CTD protein, it was shown that the latter protein exhibits highly reduced endonuclease activity. NgoL ATPase activity was enhanced in the presence of DNA. The fact that NgoL ATPase activity is stimulated ~ 2.5-fold by dsDNA and ~ 2-fold by ssDNA is a further evidence for the interaction between NgoL and DNA. The results presented above show that NgoL harbors a nicking endonuclease activity which is present in the C-terminal domain. NgoL and NgoL-CTD are dimers in solution and DMHA(X)2E(X)4E motif present in the CTD is required for the nicking endonuclease activity. These results suggest that DNA mismatch repair mechanism in N. gonorrhoeae is different from that in E. coli. In the absence of MutH homolog, N. gonorrhoeae is able to repair the DNA by virtue of MutL nicking endonuclease activity.

Des nombreux utilisateurs des systèmes multi-agents (SMA) sont très souvent découragés de modéliser et simuler dans les plates-formes actuelles SMA. Plus précisément, modéliser la dynamique d'un système (en particulier les comportements de l'agent) est très souvent vu comme un défi pour les utilisateurs de SMA. Dans le domaine des systèmes socio-écologiques (SES), cet inconvénient est plus souvent observé une fois que les experts de domaine en SES sont rarement des programmeurs. De plus, la majorité des plateformes SMA n'a pas été conçue en prenant en considérant le fait que les experts de domaines ne sont pas des programmeurs. On constate que la majeure partie des outils MAS ne sont pas dédiés à SES, ou qu'ils ne possèdent pas un formalisme compréhensible pour représenter les comportements de SMA. En outre, comme ces outils sont dépendant des plateformes, un modèle réalisé dans une plateforme SMA ne peut pas être correctement utilisé dans une autre plate-forme en raison de l'incompatibilité entre ces plateformes SMA. Afin de surpasser ces limitations, nous proposons un langage dédié au domaine SES pour décrire les comportements des agents réactifs, quelle que soit la plate-forme utilisée pour la simulation des SMA. Pour ce faire, nous avons appliqué l’approche de l’ingénierie dirigée par les modèles (IDM), une approche qui fournit des outils pour développer des langages dédiés à partir d'un méta-modèle (syntaxe abstraite), des éditeurs textuels avec coloration syntaxique (pour la syntaxe concrète) et des outils des générateurs de code (pour la génération de source code à partir d'un modèle). En conséquence, nous avons mis en œuvre un langage et un éditeur de texte qui permet à des experts du domaine SES de décrire les comportements de trois manières différentes qui sont fermées à leur expression naturelle : sous forme d'équations quand ils sont familiers avec celles-ci, en tant que séquence d'activités proche du langage naturel ou comme un diagramme d'activité pour représenter les décisions et une séquence de comportements en utilisant un formalisme graphique. Pour montrer la généralité, nous avons également développé des générateurs de code ciblant deux plates-formes différentes SMA (Cormas et Netlogo). Nous avons testé les générateurs de code en mettant en œuvre deux modèles SES avec le langage dédié développé. Le code généré obtenu a été généré pour les deux plates-formes SMA Cormas et NetLogo, et simulé avec succès dans un des deux plateformes. Nous avons conclu que l'approche IDM fournit des outils adéquats à développer des langages dédiés et des générateurs de code pour faciliter la modélisation et la simulation SMA par des non-programmeurs. En ce qui concerne le langage développé, bien que l’aspect comportemental de la simulation MAS fasse partie de la complexité de la modélisation en SMA, il y a encore d'autres aspects essentiels du modèle et de la simulation de SMA qui sont encore à être explorés, tels que l'initialisation et les points de vue sur un le monde simulé d’un modèle. / Many users of multi-agent systems (MAS) are very commonly discouraged to model and simulate using current MAS platforms. More specifically, modeling the dynamics of a system (in particular the agent's behaviors) is very often a challenge to users of MAS. That issue is more often observed in the domain of socio-ecological systems (SES), because SES domain experts are rarely programmers. Indeed,the majority of MAS platforms were not conceived taking into consideration domain-experts that are non-programmers. Most of the current MAS tools are not dedicated to SES, or they do not possess an easily understandable formalism to represent behaviors of agents. Moreover, because it is platform-dependent, a model realized in a MAS platform cannot be properly used in another platform due to incompatibility between MAS platforms. To overcome these limitations, we propose a domain-specific language (DSL) to describe the behaviors of reactive agents, regardless of the MAS platform used for simulation. To achieve that, we applied model-driven engineering (MDE), an approach that provides tools to develop DSLs from a meta-model (abstract syntax), textual editors with syntax highlighting (for the concrete syntax) and code generation capabilities (for source-code generation of a model). As a result, we implemented a language and a textual editor that allows SES domain experts to describe behaviors in three different ways that are closed to their natural expression: as equations when they are familiar to those, as a sequence of activities close to natural language or as an activity diagram to represent decisions and a sequence of behaviors using a graphic formalism. To show the generality we also developed code generators targeting two different MAS platforms (Cormas and Netlogo). We tested the code generators by implementing two SES models with the developed DSL. The generated code was targeted for both MAS platforms (Cormas and Netlogo), and successfully simulated in one of them.We conclude that the MDE approach provides adequate tools to develop DSL and code generators to facilitate MAS modeling and simulation by non-programmers. Concerning the developed DSL, although the behavioral aspect of MAS simulation is part of the complexity of modeling in MAS, there are still other essential aspects of model and simulation of MAS that are yet to be explored, such as model's initialization and points of view on the model's simulated world

No description available.

L'observation que nous avons faite d'une plus grande fréquence de souches de Staphylococcus aureus hypermutables isolées chez les patients atteints de mucoviscidose que chez un panel de souches témoins nous a amené à étudier les mécanismes de l'hypermutabilité chez cette espèce. L'analyse des gènes du système de réparation des mésappariements, mutS et mutL, a montré pour quatre souches cliniques une relation de cause à effet entre l'altération de ces gènes et leur phénotype hypermutable. Nous avons montré que ces gènes étaient co-transcrits chez S. aureus et que l'invalidation de l'un ou l'autre conduisait à un phénotype hypermutable. Nous avons mis au point un modèle pour l'étude de l'effet de ces gènes sur la recombinaison chez S. aureus, qui semble montrer que tous deux n'ont qu'un rôle limité dans le phénomène chez cette espèce. Cette propriété d'hypermutabilité a aussi un impact sur la résistance aux antibiotiques des souches. En effet, la résistance aux antibiotiques du groupe des macrolides est de plus en plus fréquente chez les souches de S. aureus isolées lors de la mucoviscidose, et nous avons montré qu'elle n'était pas due à des gènes de résistance portés par des plasmides ou des transposons mais, de façon très inhabituelle, à des mutations de la cible ribosomale des macrolides. Nous avons ainsi mis en évidence des mutations dans les gènes rrl (codant l'ARN ribosomal), rplD et rplV (codant respectivement les protéines ribosomales L4 et L22).

DNA repair processes are crucial for mutation avoidance and the maintenance of genetic integrity in all organisms. Organisms rely on repair processes to combat genotoxic stress imposed by hostile host environment, and sometimes by therapeutic agents. Most pathogens rapidly generate genetic variability to acquire increased virulence and evade host immune response. Therefore, there needs to exist a fine balance between mutation avoidance and fixation, which is perhaps regulated by repair processes. Haemophilus influenzae and Helicobacter pylori contribute significantly to morbidity and mortality caused by bacteria worldwide. H. influenzae is an obligate commensal of upper respiratory tract with the potential to cause a variety of diseases in humans like meningitis and respiratory infections. H. pylori, which inhabits the human stomach, is associated with gastric and duodenal ulcers and cancerous gastric lesions. One of the striking differences between these two genetically diverse bacterial species is the absence of recognized DNA mismatch repair (MMR) pathway homologs in H. pylori. MMR is a highly conserved post-replicative process, which corrects base pairing mismatches and small loops arising during DNA replication and recombination due to misincorporated nucleotides, insertions, and deletions. Defective MMR results in increased mutation frequency that can alter the pathogenic potential and antibiotic resistance of pathogens. MMR has been extensively studied in Escherichia coli, and requires an orchestrated function of different proteins like MutS, MutL, MutH, UvrD, SSB, RecJ, ExoVII, ExoI, ExoX, beta-clamp, DNA polymerase III and DNA ligase. A growing body of evidence suggests that bacteria other than the well-characterized E. coli paradigm differ in basic DNA repair machinery. MMR proteins involved in mismatch recognition and strand discrimination like MutS, MutL and MutH from H. influenzae have been characterized, but other downstream repair genes like UvrD helicase and exonucleases like RecJ have not been studied functionally in detail. H. pylori harbors a UvrD homolog, which shares limited homology with other UvrD proteins (29% identity with E. coli UvrD and 31 % with H. influenzae UvrD) and its cellular functions are not clear. Moreover, it is not well-understood how the activities of UvrD and RecJ proteins are regulated within these pathogens. It was, therefore, envisaged that biochemical characterization of UvrD and RecJ would lead to a better understanding of the mechanistic aspects of repair processes within these pathogens. The following sections summarize the results presented in this investigation. Functional characterization of UvrD from H. influenzae UvrD or DNA helicase II is a member of superfamily I of DNA helicases with well-documented roles in nucleotide excision repair (NER) and MMR, in addition to roles in replication and recombination. The 727-amino acid H. influenzae Rd KW20 UvrD (HiUvrD) protein was purified as an N-terminal (His)6-tagged protein to near homogeneity, and its authenticity was confirmed by peptide mass fingerprint analysis. HiUvrD displayed robust binding with single-stranded (ss) DNA as compared to double-stranded (ds) DNA. HiUvrD was found exhibit ~ 1000-fold higher affinity for ssDNA as compared to dsDNA as determined by surface plasmon resonance (SPR). In addition, to gain insights into the role of HiUvrD in replication, repair, recombination and transcription, the ability of HiUvrD to bind different DNA structures resembling intermediates of these processes was investigated using electrophoretic mobility shift assays. HiUvrD exhibited relatively high affinities for a number of branched DNA substrates and the order of affinity observed was; splayed-duplex ≥3’-flap ≥ ssDNA > 3’-overhang > four-way junction > three-way junction > nicked duplex > looped duplex ≥ duplex. Concurrent with its high affinity for ssDNA, HiUvrD exhibited a robust ssDNA-specific and Mg2+ - dependent ATPase activity. HiUvrD was able to unwind different DNA structures with varying efficiencies (3’ flap ≥ 3’-overhang > three-way junction > splayed-duplex > four-way junction > nicked > loop = duplex >>> 5’-overhang) and with a 3’-5’ polarity, which underpins its role in replication fork reversal, recombination and different DNA repair pathways. Multiple sequence alignment of HiUvrD with other helicases showed the presence highly conserved helicase motifs of which motif I and II are essential for ATP binding and hydrolysis. Mutation of an invariant glutamate residue (E226Q) in motif II of HiUvrD resulted in a dominant negative growth phenotype since, it was not possible to recover transformants when wild-type E. coli expression strains BL21(DE3)plysS or BL21(DE3)plysE were transformed with expression vector carrying hiuvrDE226Q. Mutation of a conserved arginine residue to alanine (R288A) in motif IV resulted in approximately 80 % reduction in ATP hydrolysis, and abrogation of helicase activity as compared to the wild-type protein. This can be attributed to ~ 70 % reduced ATP binding by HiUvrDR288A as determined by UV-crosslinking of radioactive ATP without change in affinity for ssDNA. HiUvrD was found to exist predominantly as a monomer with small amounts (~ 2-3 %) of higher oligomers like dimers and tetramers in solution. Deletion of 48 amino acid residues from distal C-terminus of HiUvrD resulted in abrogation of the oligomeric species implicating C-terminus to be involved in protein oligomerization. Interplay of UvrD with MutL and MutS in H. influenzae, and its modulation by ATP To investigate the effects of H. influenzae MutS (HiMutS) and MutL (HiMutL) on the helicase activity of HiUvrD, two different nicked DNA substrates were generated- a homoduplex and a heteroduplex DNA with a GT mismatch. HiMutL and HiMutS did not exhibit any helicase activity on either homoduplex or heteroduplex DNA, and unwinding of these substrates was observed only in presence of HiUvrD. In the presence of HiMutL the helicase activity of HiUvrD was stimulated on both homoduplex and heteroduplex nicked substrates whereas no significant modulation of HiUvrD ATPase activity in presence of HiMutL was observed. A much higher stimulation of unwinding of heteroduplex DNA was obtained, in presence of increasing concentrations of HiMutS. With increasing concentrations of HiMutL a progressive increase in HiUvrD mediated unwinding of the radiolabeled DNA strand was observed, which was ~ 15-fold higher than unwinding by HiUvrD alone. To investigate the effect of ATP in the stimulation of HiUvrD by HiMutL, two mutants of HiMutL–E29A (E29 is involved in ATP hydrolysis in E. coli UvrD), and D58A (D58 is essential for ATP binding in E. coli UvrD) were generated. HiMutLE29A retained only ~ 30 % of the wild-type ATPase activity, which was completely abolished in HiMutLD58A. Similar to wild-type protein, HiMutLE29A was able to stimulate HiUvrD helicase activity whereas HiMutLD58A failed to stimulate this activity. This indicated that ATP-bound form of MutL was essential for stimulation and perhaps interaction with UvrD. SPR analysis was carried out to validate and quantitate the direct protein-protein interaction between HiUvrD and HiMutL in absence or in presence of ATP, AMPPNP, and ADP. In the presence of ATP as well as AMPPNP, almost ~ 10,000-fold increase in the affinity between HiMutL and HiUvrD was observed but the same was not the case in presence of ADP. This clearly suggested that ATP binding rather than its hydrolysis promotes the interaction of MutL with UvrD. The effect of HiMutS on MutL-stimulated DNA unwinding by HiUvrD was determined using a heteroduplex nicked DNA with a GT mismatch. Interestingly, in the presence of HiMutS ~ 20-fold activation of DNA unwinding was observed, which is higher than the stimulation by HiMutL alone. The role of ATP-hydrolysis by MutS in regulation of UvrD helicase was studied by replacing wild-type protein with HiMutSE696A in the helicase assays. HiMutSE696A failed to hydrolyze ATP but was able to bind ATP with the same affinity as the wild-type protein and interacted with heteroduplex DNA with ~ 8-fold reduced affinity as compared to wild-type MutS. Intriguingly, increasing concentrations of HiMutSE696A failed to stimulate HiUvrD helicase activity in presence of HiMutL indicating that ATP hydrolysis by HiMutS is essential for stimulation of HiUvrD helicase activity post MutH-nicking during MMR. SSB, an essential component of all DNA metabolism pathways, possibly functions to stabilize the ssDNA tract generated by UvrD and exonucleases during MMR. ATPase and helicase activities of HiUvrD were inhibited by the cognate SSB protein. This inhibition could be overcome by increasing the concentration of HiUvrD helicases thus, pointing out the fact that SSB and UvrD perhaps compete with each other for ssDNA substrate. Noticeably, MutL and MutS proteins could alleviate the inhibition of HiUvrD by HiSSB. Functional characterization of UvrD from H. pylori In H. pylori, UvrD has been reported to limit homologous recombination and DNA-damage induced genomic recombinations but the protein has not been functionally studied. UvrD from H. pylori strain 26695 (HpUvrD) was over-expressed and purified as an N-terminal (His)6-tagged protein, and its authenticity was confirmed by peptide mass fingerprint analysis. HpUvrD exhibited high affinity for ssDNA as compared to dsDNA as determined by electrophoretic mobility shift assays and SPR. In addition, HpUvrD was able to bind a number of branched DNA structures (splayed duplex > ssDNA > 3’-flap > 3’overhang > three-way junction = four-way junction > loop >>> nicked ≥ duplex) suggesting its role in different DNA processing pathways. HpUvrD exhibited a Mg2+ - dependent ssDNA-specific ATPase activity, and a 3’-5’ helicase activity. HpUvrD was able to unwind different branched DNA structures with 3’-ssDNA regions like splayed duplex, 3’-overhang and 3’-flap. Blunt-ended duplex, duplexes with nick and loop as well as three-way and four-way junctions were unwound with less efficiency. Interestingly, the helicase activity of HpUvrD was supported by GTP and dGTP to almost the same level as ATP and dATP, which is in stark contrast to other characterized UvrD proteins. Moreover, HpUvrD was able to hydrolyze GTP albeit with ~ 1.5-fold reduced rate as compared to ATP. However, motifs associated with GTP binding and hydrolysis were not found in HpUvrD and it is possible that GTP binds in the same site as ATP. To investigate this possibility, helicase assay was done in the presence of ATP together with different concentrations of GMP-PNP, which is a non-hydrolysable analog of GTP, and did not support HpUvrD helicase activity. With increasing concentrations of GMP-PNP, a progressive inhibition of DNA unwinding by HpUvrD was observed suggesting that GMP-PNP could compete with ATP for a common binding site within HpUvrD. Replacement of a highly conserved glutamate residue with gluatamine (E206Q) in Walker B motif of HpUvrD resulted in ~17-fold reduced ATPase activity, and abrogation of helicase activity as compared to the wild-type protein. HpUvrDE206Q was able to bind ssDNA and ATP with comparable affinities as the wild-type protein suggesting the role of E206 in ATP hydrolysis. Like HiUvrD, HpUvrD was found to exist predominantly as a monomer in solution together with the presence of small amounts of higher oligomeric species. However, unlike HiUvrD, deletion of distal C-terminal 63 amino acids in HpUvD did not abrogate the oligomeric species suggesting that additional regions of the protein may be involved in protein oligomerization. The ATPase and helicase activities of HpUvrD were inhibited by the cognate SSB protein, and this inhibition could be overcome by increasing HpUvrD concentrations again suggesting that both UvrD and SSB proteins compete for ssDNA substrate. To investigate the role of UvrD in the physiology of H. pylori, a knock-out of hpuvrD was constructed in H. pylori strain 26695 by insertion of chloramphenicol cassette in its open reading frame. The mutant H. pylori strain 26695 obtained after disruption of hpuvrD was extremely slow growing under the normal microaerophilic conditions compared to the wild-type strain. Growth defect of H. pylori strain 26695ΔhpuvrD highlights the importance of UvrD in H. pylori cellular processes and in vitro fitness. Characterization of H. influenzae RecJ and its interaction with SSB Among the four exonucleases involved in MMR pathway, RecJ is the only known nuclease that degrades single-stranded DNA with 5’ to 3’ polarity. RecJ exonuclease plays additional important roles in base-excision repair, repair of stalled replication forks, and recombination. RecJ exonuclease from H. influenzae (HiRecJ) is a 575 amino acid protein, which harbors the characteristic motifs conserved among RecJ homologs. Due to limited solubility of HiRecJ, the protein was purified as a fusion protein with maltose binding protein (MBP). The purified protein exhibited a Mg2+ or Mn2+- dependent, and a highly processive 5’ to 3’ exonuclease activity, which is specific for ssDNA. MBP did not affect the exonuclease activity of HiRecJ. The processivity of HiRecJ was determined as ~ 700 nucleotides per binding event, using a ssDNA substrate labelled internally with 3H and at its 5’-terminus with 32P. Cd2+ inhibited the Mg2+ - dependent exonuclease activity of RecJ, which could not be overcome by increasing Mg2+ concentration. Site-directed mutagenesis of highly conserved residues in HiRecJ- D77A, D156A and H157A abolished the enzymatic activity. Interestingly, HiRecJD77A was found to interact with ssDNA with a 10-fold higher affinity than wild-type protein suggesting that this conserved aspartate residue may function to coordinate the binding of metal ion or DNA to hydrolysis of DNA. E. coli HU protein inhibited the HiRecJ exonuclease activity in a concentration-dependent manner possibly due to sequestration of ssDNA, thus making it unavailable for HiRecJ. During MMR, ssDNA tracts generated by UvrD helicase activity are most probably stabilized by SSB and hence, the in vivo substrate for RecJ would be SSB-ssDNA complex. The exonuclease activity of HiRecJ was stimulated approximately 3-fold by H. influenzae SSB (HiSSB) protein. HiSSB was able to stimulate HiRecJ exonuclease activity on a ssDNA substrate, which formed either a very strong secondary structure or on a homopolymeric ssDNA substrate, which did not form any secondary structure, suggesting that HiRecJ exonuclease was stimulated independent of the ability to HiSSB to melt secondary structures and stabilize ssDNA. Significantly, steady-state-kinetic analysis clearly showed that HiSSB increases the affinity of HiRecJ for ssDNA. H. influenzae SSBΔC and T4 gene 32 protein, a SSB homolog from bacteriophage T4, failed to enhance the HiRecJ exonuclease activity suggesting a specific functional interaction between HiSSB and HiRecJ mediated by C-terminus tail of HiSSB. More importantly, HiRecJ was found to directly associate with its cognate SSB. The C-terminus of HiSSB protein was found to be essential for this interaction. To delineate the regions of HiRecJ that interact with HiSSB, different truncated forms of HiRecJ were generated in which regions external to conserved motifs required for exonuclease activity were deleted. Different deletion mutants of HiRecJ- RecJ∆N34, RecJ∆C76 and the core catalytic domain (which contains amino acid residues 35-498) were purified as fusion proteins with MBP. HiSSB was found to interact with all the truncated forms of HiRecJ suggesting that its core-catalytic domain harbors a site for interaction with SSB. Taken together, the results presented in this study lead to a better understanding of the structure-function relationships of the UvrD helicase and RecJ exonuclease. Importantly, they provide insights into the interplay between various proteins in DNA MMR pathway. Characterization of repair proteins that are involved in multiple genome fidelity pathways is of fundamental importance to understand repair processes, more so in pathogenic bacteria wherein they regulate mutation rates, which can alter the fitness and virulence of the pathogens. Publication Sharma R., and Rao, D.N. (2009). Orchestration of Haemophilus influenzae RecJ exonuclease by interaction with single-stranded DNA-binding protein. J. Mol. Biol., 385, 1375-1396.