Catalytic and anticancer activities of sawhorse-type diruthenium tetracarbonyl complexes derived from fluorinated fatty acids

The reaction of fluorinated fatty acids, perfluorobutyric acid (C3F7CO2H), and perfluorododecanoic acid (C11F23CO2H), with dodecacarbonyltriruthenium (Ru-3(CO)(12)) under reflux in tetrahydrofuran, followed by addition of two-electron donors (L) such as pyridine, 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane, or triphenylphosphine, gives stable diruthenium complexes Ru-2(CO)(4)((2)-(2)-O2CC3F7)(2)(L)(2) (1a, L=C5H5N; 1b, L=PTA; 1c, L=PPh3) and Ru-2(CO)(4)((2)-(2)-O2CC11F23)(2)(L)(2) (2a, L=C5H5N; 2b, L=PTA; 2c, L=PPh3). The catalytic activity of the complexes for hydrogenation of styrene under supercritical carbon dioxide has been assessed and compared to the analogous triphenylphosphine complexes with non-fluorinated carboxylato groups Ru-2(CO)(4)((2)-(2)-O2CC3H7)(2)(PPh3)(2) (3) and Ru-2(CO)(4)((2)-(2)-O2CC11H23)(2)(PPh3)(2) (4). In addition, the cytotoxicities of the fluorinated complexes 1 were also evaluated on several human cancer cell lines (A2780, A549, Me300, HeLa). The complexes appear to be moderately cytotoxic, showing greater activity on the Me300 melanoma cells. Single-crystal X-ray structure analyses of 1a and 3 show the typical sawhorse-type arrangement of the diruthenium tetracarbonyl backbone with two bridging carboxylates and two terminal ligands occupying the axial positions.

Antitumor activities of ATP-competitive inhibitors of mTOR in colon cancer cells.

BACKGROUND: The mammalian target of rapamycin (mTOR) is frequently activated in colon cancers due to mutations in the phosphatidylinositol 3-kinase (PI3K) pathway. Targeting mTOR with allosteric inhibitors of mTOR such as rapamycin reduces colon cancer progression in several experimental models. Recently, a new class of mTOR inhibitors that act as ATP-competitive inhibitors of mTOR, has been developed. The effectiveness of these drugs in colon cancer cells has however not been fully characterized. METHODS: LS174T, SW480 and DLD-1 colon cancer cell lines were treated with PP242 an ATP-competitive inhibitor of mTOR, NVP-BEZ235, a dual PI3K/mTOR inhibitor or rapamycin. Tumor cell growth, proliferation and survival were assessed by MTS assay, 5-bromo-2'-deoxyuridine (BrDU) incorporation or by quantification of DNA fragmentation respectively. In vivo, the anticancer activity of mTOR inhibitors was evaluated on nude mice bearing colon cancer xenografts. RESULTS: PP242 and NVP-BEZ235 reduced the growth, proliferation and survival of LS174T and DLD-1 colon cancer cells more efficiently than rapamycin. Similarly, PP242 and NVP-BEZ235 also decreased significantly the proliferation and survival of SW480 cells which were resistant to the effects of rapamycin. In vivo, PP242 and NVP-BEZ235 reduced the growth of xenografts generated from LS174T and SW480 cells. Finally, we also observed that the efficacy of ATP-competitive inhibitors of mTOR was enhanced by U0126, a MEK inhibitor. CONCLUSIONS: Taken together, these results show that ATP-competitive inhibitors of mTOR are effective in blocking colon cancer cell growth in vitro and in vivo and thus represent a therapeutic option in colon cancer either alone or in combination with MEK inhibitors.

Cytochrome P-450 activities in human and rat brain microsomes.

The role of cytochrome P450 in the metabolism of dextromethorphan, amitriptyline, midazolam, S-mephenytoin, citalopram, fluoxetine and sertraline was investigated in rat and human brain microsomes. Depending on the parameters, the limit of quantification using gas chromatography-mass spectrometry methods was between 1.6 and 20 pmol per incubation, which generally contained 1500 microg protein. Amitriptyline was shown to be demethylated to nortriptyline by both rat and human microsomes. Inhibition studies using ketoconazole, furafylline, sulfaphenazole, omeprazole and quinidine suggested that CYP3A4 is the isoform responsible for this reaction whereas CYP1A2, CYP2C9, CYP2C19 and CYP2D6 do not seem to be involved. This result was confirmed by using a monoclonal antibody against CYP3A4. Dextromethorphan was metabolized to dextrorphan in rat brain microsomes and was inhibited by quinidine and by a polyclonal antibody against CYP2D6. Only the addition of exogenous reductase allowed the measurement of this activity in human brain microsomes. Metabolites of the other substrates could not be detected, possibly due to an insufficiently sensitive method. It is concluded that cytochrome P450 activity in the brain is very low, but that psychotropic drugs could undergo a local cerebral metabolism which could have pharmacological and/or toxicological consequences.

Gentamicin Improves the Activities of Daptomycin and Vancomycin against Enterococcus faecalis In Vitro and in an Experimental Foreign-Body Infection Model.

For enterococcal implant-associated infections, the optimal treatment regimen has not been defined. We investigated the activity of daptomycin, vancomycin, and gentamicin (and their combinations) against Enterococcus faecalis in vitro and in a foreign-body infection model. Antimicrobial activity was investigated by time-kill and growth-related heat production studies (microcalorimetry) as well as with a guinea pig model using subcutaneously implanted cages. Infection was established by percutaneous injection of E. faecalis in the cage. Antibiotic treatment for 4 days was started 3 h after infection. Cages were removed 5 days after end of treatment to determine the cure rate. The MIC, the minimal bactericidal concentration (MBC) in the logarithmic phase, and the MBC in the stationary phase were 1.25, 5, and >20 μg/ml for daptomycin, 1, >64, and >64 μg/ml for vancomycin, and 16, 32, and 4 μg/ml for gentamicin, respectively. In vitro, gentamicin at subinhibitory concentrations improved the activity against E. faecalis when combined with daptomycin or vancomycin in the logarithmic and stationary phases. In the animal model, daptomycin cured 25%, vancomycin 17%, and gentamicin 50% of infected cages. In combination with gentamicin, the cure rate for daptomycin increased to 55% and that of vancomycin increased to 33%. In conclusion, daptomycin was more active than vancomycin against adherent E. faecalis, and its activity was further improved by the addition of gentamicin. Despite a short duration of infection (3 h), the cure rates did not exceed 55%, highlighting the difficulty of eradicating E. faecalis from implants already in the early stage of implant-associated infection.

Aging and the lateralization of oscillatory activities related to external and internal motor preparation

Selection of action may rely on external guidance or be motivated internally, engaging partially distinct cerebral networks. With age, there is an increased allocation of sensorimotor processing resources, accompanied by a reduced differentiation between the two networks of action selection. The present study examines the age effects on the motor-related oscillatory patterns related to the preparation of externally and internally guided movements. Thirty-two older and 30 younger adults underwent three delayed motor tasks with S1 as preparatory and S2 as imperative cue: Full, laterality instructed by S1 (external guidance); Free, laterality freely selected (internal guidance); None, laterality instructed by S2 (no preparation). Electroencephalogram (EEG) was recorded using 64 surface electrodes. Motor-Related Amplitude Asymmetries (MRAA), indexing the lateralization of oscillatory activities, were analyzed within the S1-S2 interval in the mu (9-12 Hz) and low beta (15-20 Hz) motor-related frequency bands. Reaction times to S2 were slower in older than younger subjects, and slower in the Free than in the Full condition in older subjects only. In the Full condition, there were significant mu MRAA in both age groups, and significant low beta MRAA only in older adults. The Free condition was associated with large mu MRAA in younger adults and limited low beta MRAA in older adults. In younger subjects, the lateralization of mu activity in both Full and Free conditions indicated effective external and internal motor preparation. In older subjects, external motor preparation was associated with lateralization of low beta in addition with mu activity, compatible with an increase of motor-related resources. In contrast, absence of mu and limited low beta lateralization in internal motor preparation was concomitant with reaction time slowing and suggested less efficient cerebral processes subtending free movement selection in older adults, indicating reduced capacity for internally driven action with age.

Intraoperative angiography reloaded: a new hybrid operating theater for combined endovascular and surgical treatment of cerebral arteriovenous malformations: a pilot study on 25 patients.

BACKGROUND: Multimodality treatment suites for patients with cerebral arteriovenous malformations (AVM) have recently become available. This study was designed to evaluate feasibility, safety and impact on treatment of a new intraoperative flat-panel (FP) based integrated surgical and imaging suite for combined endovascular and surgical treatment of cerebral AVM. METHODS: Twenty-five patients with AVMs to treat with combined endovascular and surgical interventions were prospectively enrolled in this consecutive case series. The hybrid suite allows combined endovascular and surgical approaches with intraoperative scanner-like imaging (XperCT®) and intraoperative 3D rotational angiography (3D-RA). The impact of intraoperative multimodal imaging on feasibility, workflow of combined interventions, surgery, and unexpected imaging findings were analyzed. RESULTS: Twenty-five patients (mean age 38 ± 18.6 year) with a median Spetzler-Martin grade 2 AVM (range 1-4) underwent combined endovascular and surgical procedures. Sixteen patients presented with a ruptured AVM and nine with an unruptured AVM. In 16 % (n = 4) of cases, intraoperative imaging visualized AVM remnants ≤3 mm and allowed for completion of the resections in the same sessions. Complete resection was confirmed in all n = 16 patients who had follow-up angiography one year after surgery so far. All diagnostic and therapeutical steps, including angiographic control, were performed without having to move the patients CONCLUSION: The hybrid neurointerventional suite was shown to be a safe and useful setup which allowed for unconstrained combined microsurgical and neuroradiological workflow. It reduces the need for extraoperative angiographic controls and subsequent potential surgical revisions a second time, as small AVM remnants can be detected with high security.

Critical Role of the GM-CSF Signaling Pathway in Macrophage Pro-Repair Activities.

OBJECTIVE: Macrophages play a critical role in intestinal wound repair. However, the molecular pathways that regulate macrophage wound repair activities remain poorly understood. The aim of this study was to evaluate the role of GM-CSF receptor signaling in the wound repair activities of macrophages. METHODS: Murine macrophages were differentiated from bone marrow cells and human macrophages from monocytes isolated from peripheral blood mononuclear cells of Crohn's disease (CD) patients. In vitro models were used to study the repair activities of macrophages. RESULTS: We provide evidence that GM-CSF receptor signaling is required for murine macrophages to promote epithelial repair. In addition, we demonstrate that the deficient repair properties of macrophages from CD patients with active disease can be recovered via GM-CSF therapy. CONCLUSION: Our data support a critical role of the GM-CSF signaling pathway in the pro-repair activities of mouse and human macrophages. © 2014 S. Karger AG, Basel.

Das römische Theater von Avenches/Aventicum : Architektur, Baugeschichte, kulturhistorische Aspekte

Résumée Le théâtre romain d'Aventicum s'inscrit entre la petite ville moderne d'Avenches et le village de Donatyre, au pied d'une colline en pente douce délimitant au sud-est la plaine de la Broye. Il se situe à l'ouest des quartiers urbains antiques, construits selon un plan orthogonal, et s'intègre à une zone comptant divers temples et édifices publics. Dès l'hiver 1889/1890, l'Association Pro Aventico nouvellement fondée lança les premières fouilles archéologiques. Jusqu'en 1914, on dégagea les parties originales de la maçonnerie tout en assurant la restauration de l'édifice. En 1926/1927 et de 1939 à 1942 auront lieu d'autres fouilles de grande envergure, accompagnées de mesures de conservation. En 2001, la Fondation Pro Aventico lança un projet visant à étudier l'histoire de la construction ainsi que l'architecture du monument, alors connues en partie seulement. Sur la base de vestiges attestant la présence d'édifices antérieurs au théâtre, on définira pour la construction de ce dernier un terminus post quem entre 100 et 120 ap. J.-C. Comme l'indique l'étude du plan au sol, ce projet nécessita une importante planification. L'édifice lui-même se constitue d'une zone en demi-cercle réservée au public, dont les substructions indiquent qu'elle était partiellement isolée des autres. La cavea, subdivisée en trois secteurs concentriques, se termine par le bâtiment des halles et par les aditus; on relèvera que les rangées supérieures réservées aux spectateurs s'étendaient sans doute au-delà des halles et jusqu'à la façade. Les aditus permettaient d'accéder à la zone de l'orchestra et de la scène, dominée par une plate-forme de plan rectangulaire et bordée d'une proédrie. On disposait de deux voies d'accès différentes: l'une à l'avant, par les arcades des halles, et l'une à l'arrière, pratiquée dans le mur en demi-cercle; apparemment, on ne pouvait pénétrer que dans la partie centrale de ce dernier. On ne parvient à restituer que partiellement les voies de circulation dans les substructions de la cavea, en raison de leur piètre état de conservation. On a par contre pu repérer le deambulatorium, à la périphérie, ainsi que cinq vomitoria sur la première praecinctio et six vomitoria sur la seconde praecinctio. On peut admettre, sans toutefois disposer d'arguments à toute épreuve, que la troisième rangée, en haut, était accessible par des cages d'escaliers conduisant à la summa cavea. Ces hypothèses, fondées essentiellement sur le plan au sol de l'édifice et touchant aux voies de circulation, sont corroborées par une restitution des gradins des parties en élévation, aujourd'hui disparus. Quelques éléments architecturaux fournissent des arguments décisifs pour cette restitution, comme par exemple un bloc de gradin qui permet de conclure à un pendage de la cavea de 26.5°. On peut par ailleurs démontrer que le module architectural défini sur la base du plan au sol fut également appliqué lors de la planification de l'élévation. Grâce à des fragments de corniche, à deux chapiteaux de pilastre ornés de feuilles d'acanthe, à une base de pilastre engagée in situ dans la maçonnerie restaurée, et en tenant compte du module architectural, on peut proposer une reconstitution approximative de la composition de la façade de l'enceinte en demi-cercle. Si les structures architecturales révèlent que le théâtre fut planifié et édifié selon un seul et unique concept, on observe cependant quelques transformations et modifications au cours du temps. D'une part, on décèle en divers endroits des traces de réparation et de consolidation, visant sans doute à stabiliser un bâtiment ayant visiblement subi des dégâts. Par ailleurs, on a également entrepris des modifications structurelles ou fonctionnelles, comme l'édification ultérieure du postscaenium le long du mur de scène extérieur. Dans un contexte identique, on relèvera également deux murs flanquant les basiliques, qu'on suppose être en relation avec l'agrandissement du complexe architectural du temple du Cigognier et du théâtre, augmenté des deux temples édifiés au milieu du 2e s. ap. J.-C. au lieu-dit Au Lavoëx. L'excavation, au cours du dernier tiers du IIIe siècle ap. J.-C., d'un fossé de près de 6 m de large pour 1.5 m de profondeur tout autour de l'édifice fit du théâtre un véritable lieu fortifié. Au-dessus du fossé, on a pu relever une séquence stratigraphique témoignant d'une activité d'habitation à proximité du théâtre pour la période allant du IVe au VIIe siècle ap. J.-C. Il s'agit de l'un des rares cas où l'on peut, à Avenches, évoquer la présence d'un habitat de la période du Haut Moyen Age.

Genetic polymorphisms and drug interactions modulating CYP2D6 and CYP3A activities have a major effect on oxycodone analgesic efficacy and safety

Background and purpose: The major drug-metabolizing enzymes for the oxidation of oxycodone are CYP2D6 and CYP3A. A high interindividual variability in the activity of these enzymes because of genetic polymorphisms and/or drug-drug interactions is well established. The possible role of an active metabolite in the pharmacodynamics of oxycodone has been questioned and the importance of CYP3A-mediated effects on the pharmacokinetics and pharmacodynamics of oxycodone has been poorly explored. Experimental approach: We conducted a randomized crossover (five arms) double-blind placebo-controlled study in 10 healthy volunteers genotyped for CYP2D6. Oral oxycodone (0.2 mg·kg−1) was given alone or after inhibition of CYP2D6 (with quinidine) and/or of CYP3A (with ketoconazole). Experimental pain (cold pressor test, electrical stimulation, thermode), pupil size, psychomotor effects and toxicity were assessed. Key results: CYP2D6 activity was correlated with oxycodone experimental pain assessment. CYP2D6 ultra-rapid metabolizers experienced increased pharmacodynamic effects, whereas cold pressor test and pupil size were unchanged in CYP2D6 poor metabolizers, relative to extensive metabolizers. CYP2D6 blockade reduced subjective pain threshold (SPT) for oxycodone by 30% and the response was similar to placebo. CYP3A4 blockade had a major effect on all pharmacodynamic assessments and SPT increased by 15%. Oxymorphone Cmax was correlated with SPT assessment (ρS= 0.7) and the only independent positive predictor of SPT. Side-effects were observed after CYP3A4 blockade and/or in CYP2D6 ultra-rapid metabolizers. Conclusions and implications: The modulation of CYP2D6 and CYP3A activities had clear effects on oxycodone pharmacodynamics and these effects were dependent on CYP2D6 genetic polymorphism.

The alarmin HMGB1 enhances CXCR4-induced activities by binding CXCL12

A variety of chemokines and inflammatory molecules are concomitantly produced at target sites of leukocyte trafficking and homing, accounting for the complex cellular responses occurring in homeostasis and inflammation. The chemokine CXCL12 plays an essential and unique role in homeostatic regulation of leukocyte traffic and tissue regeneration. The chromatin protein HMGB1 is released by dying and distressed cells, and acts as a Damage Associated Molecular Pattern or alarmin, promoting cell migration towards the site of tissue damage. We show here that HMGB1 synergises with CXCL12 by forming a heterocomplex that we characterized by NMR chemical shift mapping. The heterocomplex enhances CXCR4-induced responses on cells of the immune system, acting exclusively through the CXCL12 receptor CXCR4, and not through the HMGB1 receptors RAGE, TLR2 and TLR4. FRET analysis show that CXCL12 and CXCL12+HMGB1 promote a different conformational change in the homodimer CXCR4. The enhancement induced by HMGB1 on CXCL12-induced migration is selective, since little changes in migration of neutrophils and PreB 300.19-CCR2+ or -CCR7+ are observed towards CXCL8 and CCR2 or CCR7 agonists. HMGB1 also promotes CXCL 12 release, which is ultimately responsible for the chemoattractant activities of HMGB1. This study highlights the role of HMGB1 in promoting CXCL12-dependent cell migration, and suggests a cooperative role of these two molecules in tissue repair as well as in pathological conditions, such as rheumatoid arthritis.

Antitumor activities of ATP-competitive inhibitors of mTOR in colon cancer cells

Le cancer colorectal est la 3ème cause de décès liée au cancer dans l'Europe de l'Ouest et nécessite une prise en charge pluridisciplinaire. Les thérapies anticancéreuses récentes développées visent à inhiber les voies de signalisation cellulaires responsables de la prolifération des cellules tumorales. L'inhibition de la voie de signalisation cellulaire mTOR, est une stratégie prometteuse. En effet, mTOR est souvent suractivé dans les cellules du cancer colorectal et régule la croissance, la prolifération et la survie cellulaire. De nombreuses études récentes ont démontrés l'importance de l'activité de mTOR dans le développement du cancer colorectal et l'efficacité anti-tumorale des inhibiteurs allostériques de mTOR, telle que la rapamycine. Récemment, une nouvelle classe d'inhibiteur de mTOR, notamment PP242 et NVP-BEZ235, agissant comme inhibiteur ATP- compétitif a été développée. L'efficacité de ces inhibiteurs n'a pas été démontrée dans le contexte du cancer colorectal. Dans cette étude, nous avons comparé l'effet de PP242, un inhibiteur ATP-compétitif de mTOR et NVP-BEZ235, un inhibiteur dual de PI3K/mTOR par rapport à la rapamycine. Nous avons étudié, in vitro, leur effet sur la croissance, la prolifération et la survie cellulaire sur des lignées cellulaires du cancer du colon (LS174, SW480 et DLD-1) et, in vivo, sur la croissance de xénogreffes dans un modèle murin. Nous avons émis l'hypothèse que l'effet des ces nouveaux inhibiteurs seraient plus importants qu'avec la rapamycine. Nous avons observé que le PP242 et le NVP-BEZ235 réduisent significativement et de façon plus marquée que la rapamycine la croissance, la prolifération et la survie cellulaire des cellules LS174T et DLD-1. Ces inhibiteurs réduisent également la prolifération et la survie cellulaire des cellules SW480 alors que celles-ci étaient résistantes à la rapamycine. Nous avons également observé que les inhibiteurs PP242 et NVP-BEZ235 réduisaient la croissance des xénogreffes avec les lignées cellulaires LS174 et SW480. Finalement, nous avons remarqué que l'effet anti-tumoral des inhibiteurs ATP-compétitifs de mTOR était potentialisé par l'U0126, un inhibiteur de MEK/MAPK, souvent activé dans les voies de signalisation cellulaire du cancer colorectal. En conclusion, nous avons observé que les inhibiteurs ATP-compétitifs de mTOR bloquent la croissance de cellules tumorales du cancer colorectal in vitro et in vivo. Ces résultats démontrent que ces inhibiteurs représentent une option thérapeutique prometteuse dans le traitement du cancer colorectal et méritent d'être évalués dans des études cliniques.

Genetic polymorphisms and drug interactions modulating CYP2D6 and CYP3A activities have a major effect on oxycodone analgesic efficacy and safety.

BACKGROUND AND PURPOSE: The major drug-metabolizing enzymes for the oxidation of oxycodone are CYP2D6 and CYP3A. A high interindividual variability in the activity of these enzymes because of genetic polymorphisms and/or drug-drug interactions is well established. The possible role of an active metabolite in the pharmacodynamics of oxycodone has been questioned and the importance of CYP3A-mediated effects on the pharmacokinetics and pharmacodynamics of oxycodone has been poorly explored. EXPERIMENTAL APPROACH: We conducted a randomized crossover (five arms) double-blind placebo-controlled study in 10 healthy volunteers genotyped for CYP2D6. Oral oxycodone (0.2 mg x kg(-1)) was given alone or after inhibition of CYP2D6 (with quinidine) and/or of CYP3A (with ketoconazole). Experimental pain (cold pressor test, electrical stimulation, thermode), pupil size, psychomotor effects and toxicity were assessed. KEY RESULTS: CYP2D6 activity was correlated with oxycodone experimental pain assessment. CYP2D6 ultra-rapid metabolizers experienced increased pharmacodynamic effects, whereas cold pressor test and pupil size were unchanged in CYP2D6 poor metabolizers, relative to extensive metabolizers. CYP2D6 blockade reduced subjective pain threshold (SPT) for oxycodone by 30% and the response was similar to placebo. CYP3A4 blockade had a major effect on all pharmacodynamic assessments and SPT increased by 15%. Oxymorphone C(max) was correlated with SPT assessment (rho(S)= 0.7) and the only independent positive predictor of SPT. Side-effects were observed after CYP3A4 blockade and/or in CYP2D6 ultra-rapid metabolizers. CONCLUSIONS AND IMPLICATIONS: The modulation of CYP2D6 and CYP3A activities had clear effects on oxycodone pharmacodynamics and these effects were dependent on CYP2D6 genetic polymorphism.

Biochemical properties of RuvBD113N: a mutation in helicase motif II of the RuvB hexamer affects DNA binding and ATPase activities.

Many DNA helicases utilise the energy derived from nucleoside triphosphate hydrolysis to fuel their actions as molecular motors in a variety of biological processes. In association with RuvA, the E. coli RuvB protein (a hexameric ring helicase), promotes the branch migration of Holliday junctions during genetic recombination and DNA repair. To analyse the relationship between ATP-dependent DNA helicase activity and branch migration, a site-directed mutation was introduced into the helicase II motif of RuvB. Over-expression of RuvBD113N in wild-type E. coli resulted in a dominant negative UVs phenotype. The biochemical properties of RuvBD113N were examined and compared with wild-type RuvB in vitro. The single amino acid substitution resulted in major alterations to the biochemical activities of RuvB, such that RuvBD113N was defective in DNA binding and ATP hydrolysis, while retaining the ability to form hexameric rings and interact with RuvA. RuvBD113N formed heterohexamers with wild-type RuvB, and could inhibit RuvB function by affecting its ability to bind DNA. However, heterohexamers exhibited an ability to promote branch migration in vitro indicating that not all subunits of the ring need to be catalytically competent.

Escherichia coli RuvBL268S: a mutant RuvB protein that exhibits wild-type activities in vitro but confers a UV-sensitive ruv phenotype in vivo.

The RuvABC proteins of Escherichia coli process recombination intermediates during genetic recombination and DNA repair. RuvA and RuvB promote branch migration of Holliday junctions, a process that extends heteroduplex DNA. Together with RuvC, they form a RuvABC complex capable of Holliday junction resolution. Branch migration by RuvAB is mediated by RuvB, a hexameric ring protein that acts as an ATP-driven molecular pump. To gain insight into the mechanism of branch migration, random mutations were introduced into the ruvB gene by PCR and a collection of mutant alleles were obtained. Mutation of leucine 268 to serine resulted in a severe UV-sensitive phenotype, characteristic of a ruv defect. Here, we report a biochemical analysis of the mutant protein RuvBL268S. Unexpectedly, the purified protein is fully active in vitro with regard to its ATPase, DNA binding and DNA unwinding activities. It also promotes efficient branch migration in combination with RuvA, and forms functional RuvABC-Holliday junction resolvase complexes. These results indicate that RuvB may perform some additional, and as yet undefined, function that is necessary for cell survival after UV-irradiation.