Gotowa bibliografia na temat „UvrD helicase”

ABSTRACT Nucleotide excision repair (NER) is universally used to recognize and remove many types of DNA damage. In eubacteria, the NER system typically consists of UvrA, UvrB, UvrC, the UvrD helicase, DNA polymerase I, and ligase. In addition, when DNA damage blocks transcription, transcription-repair coupling factor (TRCF), the product of the mfd gene, recruits the Uvr complex to repair the damage. Previous work using selected mutants and assays have indicated that pathogenic Neisseria spp. carry a functional NER system. In order to comprehensively examine the role of NER in Neisseria gonorrhoeae DNA recombination and repair processes, the predicted NER genes (uvrA, uvrB, uvrC, uvrD, and mfd) were each disrupted by a transposon insertion, and the uvrB and uvrD mutants were complemented with a copy of each gene in an ectopic locus. Each uvr mutant strain was highly sensitive to UV irradiation and also showed sensitivity to hydrogen peroxide killing, confirming that all of the NER genes in N. gonorrhoeae are functional. The effect of RecA expression on UV survival was minor in uvr mutants but much larger in the mfd mutant. All of the NER mutants demonstrated wild-type levels of pilin antigenic variation and DNA transformation. However, the uvrD mutant exhibited higher frequencies of PilC-mediated pilus phase variation and spontaneous mutation, a finding consistent with a role for UvrD in mismatch repair. We conclude that NER functions are conserved in N. gonorrhoeae and are important for the DNA repair capabilities of this strict human pathogen.

ABSTRACT DNA polymerase I (PolI) functions both in nucleotide excision repair (NER) and in the processing of Okazaki fragments that are generated on the lagging strand during DNA replication.Escherichia coli cells completely lacking the PolI enzyme are viable as long as they are grown on minimal medium. Here we show that viability is fully dependent on the presence of functional UvrA, UvrB, and UvrD (helicase II) proteins but does not require UvrC. In contrast, ΔpolA cells grow even better when theuvrC gene has been deleted. Apparently UvrA, UvrB, and UvrD are needed in a replication backup system that replaces the PolI function, and UvrC interferes with this alternative replication pathway. With specific mutants of UvrC we could show that the inhibitory effect of this protein is related to its catalytic activity that on damaged DNA is responsible for the 3′ incision reaction. Specific mutants of UvrA and UvrB were also studied for their capacity to support the PolI-independent replication. Deletion of the UvrC-binding domain of UvrB resulted in a phenotype similar to that caused by deletion of the uvrC gene, showing that the inhibitory incision activity of UvrC is mediated via binding to UvrB. A mutation in the N-terminal zinc finger domain of UvrA does not affect NER in vivo or in vitro. The same mutation, however, does give inviability in combination with the ΔpolA mutation. Apparently the N-terminal zinc-binding domain of UvrA has specifically evolved for a function outside DNA repair. A model for the function of the UvrA, UvrB, and UvrD proteins in the alternative replication pathway is discussed.

Escherichia coli UvrD is a superfamily 1 helicase/translocase that functions in DNA repair, replication, and recombination. Although a UvrD monomer can translocate along single-stranded DNA, self-assembly or interaction with an accessory protein is needed to activate its helicase activity in vitro. Our previous studies have shown that an Escherichia coli MutL dimer can activate the UvrD monomer helicase in vitro, but the mechanism is not known. The UvrD 2B subdomain is regulatory and can exist in extreme rotational conformational states. By using single-molecule FRET approaches, we show that the 2B subdomain of a UvrD monomer bound to DNA exists in equilibrium between open and closed states, but predominantly in an open conformation. However, upon MutL binding to a UvrD monomer–DNA complex, a rotational conformational state is favored that is intermediate between the open and closed states. Parallel kinetic studies of MutL activation of the UvrD helicase and of MutL-dependent changes in the UvrD 2B subdomain show that the transition from an open to an intermediate 2B subdomain state is on the pathway to helicase activation. We further show that MutL is unable to activate the helicase activity of a chimeric UvrD containing the 2B subdomain of the structurally similar Rep helicase. Hence, MutL activation of the monomeric UvrD helicase is regulated specifically by its 2B subdomain.

Escherichia coli UvrD DNA helicase functions in several DNA repair processes. As a monomer, UvrD can translocate rapidly and processively along ssDNA; however, the monomer is a poor helicase. To unwind duplex DNA in vitro, UvrD needs to be activated either by self-assembly to form a dimer or by interaction with an accessory protein. However, the mechanism of activation is not understood. UvrD can exist in multiple conformations associated with the rotational conformational state of its 2B subdomain, and its helicase activity has been correlated with a closed 2B conformation. Using single-molecule total internal reflection fluorescence microscopy, we examined the rotational conformational states of the 2B subdomain of fluorescently labeled UvrD and their rates of interconversion. We find that the 2B subdomain of the UvrD monomer can rotate between an open and closed conformation as well as two highly populated intermediate states. The binding of a DNA substrate shifts the 2B conformation of a labeled UvrD monomer to a more open state that shows no helicase activity. The binding of a second unlabeled UvrD shifts the 2B conformation of the labeled UvrD to a more closed state resulting in activation of helicase activity. Binding of a monomer of the structurally similar Escherichia coli Rep helicase does not elicit this effect. This indicates that the helicase activity of a UvrD dimer is promoted via direct interactions between UvrD subunits that affect the rotational conformational state of its 2B subdomain.

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

ABSTRACT The uvrD gene in Escherichia coli encodes a 720-amino-acid 3′-5′ DNA helicase which, although nonessential for viability, is required for methyl-directed mismatch repair and nucleotide excision repair and furthermore is believed to participate in recombination and DNA replication. We have shown in this study that null mutations in uvrD are incompatible withlon, the incompatibility being a consequence of the chronic induction of SOS in uvrD strains and the resultant accumulation of the cell septation inhibitor SulA (which is a normal target for degradation by Lon protease). uvrD-lonincompatibility was suppressed by sulA,lexA3(Ind−), or recA (Def) mutations. Other mutations, such as priA, dam,polA, and dnaQ (mutD) mutations, which lead to persistent SOS induction, were also lonincompatible. SOS induction was not observed in uvrC andmutH (or mutS) mutants defective, respectively, in excision repair and mismatch repair. Nor wasuvrD-mediated SOS induction abolished by mutations in genes that affect mismatch repair (mutH), excision repair (uvrC), or recombination (recB andrecF). These data suggest that SOS induction inuvrD mutants is not a consequence of defects in these three pathways. We propose that the UvrD helicase participates in DNA replication to unwind secondary structures on the lagging strand immediately behind the progressing replication fork, and that it is the absence of this function which contributes to SOS induction inuvrD strains.

ABSTRACT The role of the C terminus of Escherichia coli DNA helicase II (UvrD), a region outside the conserved helicase motifs, was investigated by using three mutants: UvrDΔ107C (deletion of the last 107 C-terminal amino acids), UvrDΔ102C, and UvrDΔ40C. This region, which lacks sequence similarity with other helicases, may function to tailor UvrD for its specific in vivo roles. Genetic complementation assays demonstrated that mutant proteins UvrDΔ107C and UvrDΔ102C failed to substitute for the wild-type protein in methyl-directed mismatch repair and nucleotide excision repair. UvrDΔ40C protein fully complemented the loss of helicase II in both repair pathways. UvrDΔ102C and UvrDΔ40C were purified to apparent homogeneity and characterized biochemically. UvrDΔ102C was unable to bind single-stranded DNA and exhibited a greatly reduced single-stranded DNA-stimulated ATPase activity in comparison to the wild-type protein (k cat = 0.01% of the wild-type level). UvrDΔ40C was slightly defective for DNA binding and was essentially indistinguishable from wild-type UvrD when single-stranded DNA-stimulated ATP hydrolysis and helicase activities were measured. These results suggest a role for a region near the C terminus of helicase II in binding to single-stranded DNA.

G-quadruplex (G4) secondary structures have been implicated in various biological processes, including gene expression, DNA replication and telomere maintenance. However, unresolved G4 structures impede replication progression which can lead to the generation of DNA double-strand breaks and genome instability. Helicases have been shown to resolve G4 structures to facilitate faithful duplication of the genome. Escherichia coli UvrD (EcUvrD) helicase plays a crucial role in nucleotide excision repair, mismatch repair and in the regulation of homologous recombination. Here, we demonstrate a novel role of E. coli and Neisseria gonorrhoeae UvrD in resolving G4 tetraplexes. EcUvrD and N. gonorrhoeae UvrD were proficient in unwinding previously characterized tetramolecular G4 structures. Notably, EcUvrD was equally efficient in resolving tetramolecular and bimolecular G4 DNA that were derived from the potential G4-forming sequences from the genome of E. coli. Interestingly, in addition to resolving intermolecular G4 structures, EcUvrD was robust in unwinding intramolecular G4 structures. These data for the first time provide evidence for the role of UvrD in the resolution of G4 structures, which has implications for the in vivo role of UvrD helicase in G4 DNA resolution and genome maintenance.

Abstract The recJ gene encodes a single-strand DNA-specific exonuclease involved in homologous recombination. We have isolated a pseudorevertant strain in which recJ mutant phenotypes were alleviated. Suppression of recJ was due to at least three mutations, two of which we have identified as alterations in DNA helicase genes. A recessive amber mutation, "uvrD517am," at codon 503 of the gene encoding helicase II was sufficient to suppress recJ partially. The uvrD517am mutation does not eliminate uvrD function because it affects UV survival only weakly; moreover, a uvrD insertion mutation could not replace uvrD517am as a suppressor. However, suppression may result from differential loss of uvrD function: mutation rate in a uvrD517am derivative was greatly elevated, equal to that in a uvrD insertion mutant. The second cosuppressor mutation is an allele of the helD gene, encoding DNA helicase IV, and could be replaced by insertion mutations in helD. The identity of the third cosuppressor "srjD" is not known. Strains carrying the three cosuppressor mutations exhibited hyperrecombinational phenotypes including elevated excision of repeated sequences. To explain recJ suppression, we propose that loss of antirecombinational helicase activity by the suppressor mutations stabilizes recombinational intermediates formed in the absence of recJ.

Ce travail s'intéresse au rôle de l'hélicase UvrD dans le redémarrage des fourches de réplication inactivées chez Escherichia coli. Dans le cas d'arrêt de la réplication à un site Ter/Tus de terminaison de la réplication ectopique, nous avons montré que UvrD est l'hélicase majeure impliquée dans le redémarrage. Nous proposons que le rôle de l'hélicase UvrD lors du redémarrage des fourches bloquées à un site de terminaison ectopique consiste à déplacer le complexe Ter/Tus de terminaison lors du redémarrage de la réplication après recombinaison homologue. Nous avons pu montrer que ce rôle de UvrD est conservé chez son homologue PcrA de Bacillus subtilis. Dans le cas d'arrêt de la réplication après inactivation d'une sous-unité de la polymérase réplicative (Pol III) d'E. Coli, la sous-unité catalytique (mutant dnaEts) ou le facteur de processivité (mutant dnaNts), l'action anti-RecA de UvrD est essentielle à la réaction de réversion des fourches de réplication (RPR) permettant un redémarrage de la réplication. L'étude du mode d'action anti-RecA de UvrD aux fourches de réplication inactivées montre qu'il dépend de la sous-unité de Pol III inactivée. Dans le mutant dnaNts l'activité ATPase de UvrD est essentielle à son action anti-RecA. Au contraire, dans le mutant dnaEts l'action anti-RecA de UvrD ne nécessite pas l'activité ATPase de la protéine, et corrèle avec un besoin de la protéine RarA/MgsA pour la fixation de RecA. Par ailleurs, nous avons montré que l'action anti-RecA de UvrD aux fourches de réplication inactivées est conservé chez son homologue PcrA de Bacillus subtilis
This study aims at understanding the role of the UvrD helicase in the restart of arrested replication forks in Escherichia coli. After replication arrest at ectopic replication terminaison sites of Ter/Tus, we showed that UvrD is the major helicase needed to restart. We propose that UvrD acts in concert with homologous recombination proteins to dislodge Tus form Ter sites during replication restart. The Tus-removal action of UvrD is conserved in Bacillus subtilis homologous helicase PcrA. After replication fork arrest by the inactivation of a subunit of the DNA polymerase III holoenzyme (Pol Illh), either the catalytic subunit (dnaEts mutant) or the (3-clamp (dnaNts mutant), the anti-RecA action of UvrD at blocked forks is essential for the replication fork reversal reaction (RPR) to promote replication restart. We have shown that the anti-RecA action of UvrD at blocked forks reflects two different activities of this enzyme. An ATPase-deficient UvrD mutant is able to antagonize RecA in cells affected for the Pol IIIh catalytic subunit DnaE. In this mutant, RecA action at blocked forks specifically requires the protein RarA (MgsA). This suggests that UvrD acts by preventing RecA binding, possibly through counteracting RarA. In contrast, at forks affected for the Pol Illh clamp (DnaN), RarA is not required for RecA binding and the ATPase fonction of UvrD is essential to counteract RecA, supporting the idea that UvrD removes RecA from DNA. The anti-RecA action of UvrD at Pol IIIts-blocked forks is conserved in the Bacillus subtilis homologous helicase PcrA. Proliferative disorders, this unique mutation will permit a new molecular classification of these disorders and novel therapeutical approaches

A trinucleotide repeat (TNR) is a 3 base pair (bp) DNA sequence tandemly repeated in an array. In humans, TNR sequences have been found to be associated with at least 14 severe neurological diseases including Huntington disease, myotonic dystrophy and several of the spinocerebellar ataxias. Such diseases are caused by an expansion of the repeat sequence beyond a threshold length and are characterized by non-Mendelian patterns of inheritance which lead to genetic anticipation. Although the mechanism of the genetic instability in these arrays is not yet fully understood, various models have been suggested based on the in vitro observation that TNR sequences can form secondary structures such as pseudo-hairpins. In order to investigate the mechanisms responsible for instability of TNR sequences, a study was carried out on Escherichia coli cells in which TNR arrays had been integrated into the chromosomal lacZ gene. This genetic assay was used to identify proteins and pathways involved in deletion and/or expansion instability. Deletion instability was clearly dependent on orientation of the TNR sequence relative to the origin of replication. Interestingly, it was found that expansion instability is not dependent on the orientation of the repeat array relative to the origin of replication. The replication fork reversal pathway and the RecFOR mediated gap repair pathway were found to have no statistically significant influence on the instability of TNR arrays. However, the protein UvrD was found to affect the deletion instability of TNR sequences. The roles of key helicase genes were investigated for their effects on instability of chromosomal CTG•CAG repeats. Mutation of the rep gene increased deletion in the CTG leading-strand orientation of the repeat array, and expansion in both orientations - destabilizing the TNR array. RecQ helicase was found to have a significant effect on TNR instability in the orientation in which CAG repeats were present on the leading-strand relative to the origin of replication. Mutation of the recQ gene severely limited the number of expansion events in this orientation, whilst having no effect on deletions. This dependence of expansions on RecQ was lost in a rep mutant strain. In a rep mutant expansions were shown to be partially dependent on the DinG helicase. All together, these results suggest a model of TNR instability in which expansions are due to events occurring at either the leading or lagging strand of an arrested replication fork, facilitated by helicase action. The identity of the helicase implicated is determined by the nature of the arrest.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.
Title from electronic title page (viewed Feb. 17, 2009). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biology." Discipline: Biology; Department/School: Biology.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.
Title from electronic title page (viewed Feb. 17, 2009). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry." Discipline: Chemistry; Department/School: Chemistry.

G-quadruplex (G4) secondary structures have been implicated in various biological processes, including gene expression, DNA replication and telomere maintenance. However, unresolved G4 structures impede replication progression which can lead to the generation of DNA double-strand breaks and genome instability. Helicases have been shown to resolve G4 structures to facilitate faithful duplication of the genome. Escherichia coli UvrD (EcUvrD) helicase plays a crucial role in nucleotide excision repair, mismatch repair and in the regulation of homologous recombination. Here, we demonstrate a novel role of E. coli and Neisseria gonorrhoeae UvrD in resolving G4 tetraplexes. EcUvrD and N. gonorrhoeae UvrD were proficient in unwinding previously characterized tetramolecular G4 structures. Notably, EcUvrD was equally efficient in resolving tetramolecular and bimolecular G4 DNA that were derived from the potential G4-forming sequences from the genome of E. coli. Interestingly, in addition to resolving intermolecular G4 structures, EcUvrD was robust in unwinding intramolecular G4 structures. These data for the first time provide evidence for the role of UvrD in the resolution of G4 structures, which has implications for the in vivo role of UvrD helicase in G4 DNA resolution and genome maintenance.

UvrD helicase belongs to type I superfamily of helicases. It participates in Nucleotide Excision Repair (NER) and DNA Mismatch Repair (MMR). It also participates in the rolling circle replication. It has been shown that UvrD can dismantle several proteins from DNA such as RecA and Tus protein. By unwinding the recombination intermediates and dismantling RecA from DNA, it acts as an anti- recombinase. UvrD has also been shown to physically interact with RNA polymerase and helps in the transcription coupled repair. In addition to these roles, UvrD helicase also contributes to the virulence of many pathogenic bacteria. In Mycobacterium tuberculosis, the inactivation of uvrD1 gene reduced its persistence in a mouse model of tuberculosis infection. Inactivation of uvrD gene in Neisseria meningitis increases the rate of phase variation. The uvrD null mutants of Haemophilus influenza exhibit a high degree of UV sensitivity and reduced level of host cell reactivation and decreased phage recombination. Biochemical studies of Helicobacter pylori UvrD (HpUvrD) shows that it can unwind DNA duplex using the energy derived from GTP hydrolysis and deletion of the C- terminal 63 residues disrupted the oligomerisation of HpUvrD. (30). Similar studies with Haemophilus influenzae UvrD reveal that the C- terminal 48 residues are important for its oligomerisation (30). In spite of these important roles in the virulence of pathogenic bacteria, a few UvrD helicase from pathogenic bacteria have been bio-chemically characterized. In this study, UvrD helicase from N. gonorrohoeae (NgoUvrD) has been bio-chemically characterized. NgoUvrD was cloned into pET14b vector between NdeI and BamHI. The protein was heterologously expressed as N- terminal His6 tag. NgoUvrD was then purified with Ni2+-NTA affinity chromatography followed by Heparin Sepharose chromatography. Size exclusion chromatography indicates that NgoUvrD behaves as dimer in solution. NgoUvrD has been found to unwind varied range of substrates like DNA with 5’ overhang, DNA with 3’ overhang, Holliday junction and blunt end duplex DNA. However, for blunt end duplex DNA the unwinding efficiency of NgoUvrD decreases with the length of DNA. Moreover, streptavidin displacement assay demonstrates that NgoUvrD translocates in 3’ to 5’ direction. Despite this how it can unwind DNA substrates with 5’ or 3’ overhangs with equal efficiency is yet to be studied.

Homologous recombination is generally considered a major mechanism by which cells repair many types of DNA lesions and damaged replication forks. However, if this process is left unchecked, cells often show a hyper-recombination (hyper-rec) phenotype, and are susceptible to large deletions, duplications, or inversions of important genetic information. This dissertation describes two projects aimed at examining molecular mechanisms by which cells regulate homologous recombination. The first shows several 3'-5' exonucleases prevent RecA-GFP loading by destroying potential substrates. It is shown that two genetic pathways exist: one consisting of ExoIII and another comprised of ExoVII, ExoIX, ExoX, and ExoXI. ExoI acts upstream of both of these pathways. Although xthA cells have an increase in DSBs and recB-dependent loading of RecA-GFP, they are viable with a recB mutation and do not display a large increase in SOS expression. The increase in RecA-GFP is also independent of base excision repair (BER). These experiments uncovered that DNA in a population of wild type cells undergoes DSBs and is often repaired in a RecA-independent manner after processing by ExoI and ExoIII. The second project shows the helicase, UvrD limits the number and intensities of RecA-GFP foci. This activity is due to the ability of UvrD to remove RecA from DNA where it is loaded in a RecF pathway-dependent manner. This activity requires ATP binding by UvrD, suggesting that helicase/translocase activity is important for RecA-removal. The hyper-helicase mutation, uvrD303 confers UV sensitivity to cells. Epistasis analyses showed uvrD303 is defective in the recA pathway of UV repair and not in nucleotide excision repair (NER). Surprisingly, UvrD303 does not directly remove RecA after UV, as new RecA-GFP foci appear like in wild type cells. UvrD303 does, however, slightly inhibit SOS induction, and constitutively activating the SOS response restores UV resistance to these cells in a way that is independent of recA overexpression. Furthermore, uvrD303 was capable of suppressing the constitutive SOS phenotype of recA730. These experiments suggested that UvrD303 antagonizes the ability of RecA filaments to induce the SOS response, rendering cells UV sensitive.

Unresolved G-quadruplex (G4) DNA secondary structures impede DNA replication and can lead to DNA breaks and to genome instability. Helicases are known to unwind G4 structures and thereby facilitate genome duplication. Escherichia coli UvrD is a multifunctional helicase that participates in DNA repair, recombination and replication. Previously, we had demonstrated a novel role of E. coli UvrD helicase in resolving G4 structures. Mycobacterium tuberculosis genome encodes two orthologs of E. coli UvrD helicase, UvrD1 and UvrD2. It is unclear whether UvrD1 or UvrD2 or both helicases unwind G4 DNA structures. Here, we demonstrate that M. tuberculosis UvrD1 and UvrD2 unwind G4 tetraplexes. Both helicases were proficient in resolving previously characterized tetramolecular G4 structures in an ATP hydrolysis and single-stranded 3′-tail-dependent manner. Notably, M. tuberculosis UvrD1 and UvrD2 were efficient in unwinding G4 structures derived from the potential G4 forming sequences present in the M. tuberculosis genome. These data suggest an extended role for M. tuberculosis UvrD1 and UvrD2 helicases in resolving G4 DNA structures and provide insights into the maintenance of genome integrity via G4 DNA resolution.

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.

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.