Mechanism Of DNA Unwinding Research Articles

Intermediates revealed in the kinetic mechanism for DNA unwinding by a monomeric helicase

Helicases unwind dsDNA during replication, repair and recombination in an ATP-dependent reaction. The mechanism for helicase activity can be studied using oligonucleotide substrates to measure formation of single-stranded (ss) DNA from double-stranded (ds) DNA. This assay provides an 'all-or-nothing' readout because partially unwound intermediates are not detected. We have determined conditions under which an intermediate in the reaction cycle of Dda helicase can be detected by trapping a partially unwound substrate. The appearance of this intermediate supports a model in which each ssDNA product interacts with the helicase after unwinding has occurred. Kinetic analysis indicates that the intermediate appears during a slow step in the reaction cycle that is flanked by faster steps for unwinding. These observations demonstrate a complex mechanism containing nonuniform steps for a monomeric helicase. The potential biological significance of such a mechanism is discussed.

Mechanism of Translocation and Kinetics of DNA Unwinding by the Helicase RecG

RecG is a DNA helicase involved in the repair of damage at a replication fork and catalyzes the reversal of the fork to a point beyond the damage in the template strand. It unwinds duplex DNA in reactions that are coupled to ATP hydrolysis. The kinetic mechanism of duplex DNA unwinding by RecG was analyzed using a quantitative fluorescence assay based on the process of contact quenching between Cy3 and Dabcyl groups attached to synthetic three-way DNA junctions. The data show that the protein moves at a rate of 26 bp s(-1) along the duplex DNA during the unwinding process. RecG ATPase activity during translocation indicates a constant rate of 7.6 s(-1), measured using a fluorescent phosphate sensor, MDCC-PBP. These two rates imply a movement of approximately 3 bp per ATP hydrolyzed. We demonstrate in several trapping experiments that RecG remains attached to DNA after translocation to the end of the arm of the synthetic DNA junction. ATPase activity continues after translocation is complete. Dissociation of RecG from the product DNA occurs only very slowly, suggesting strong interactions between them. The data support the idea that interactions of the duplex template arm with the protein are the major sites of binding and production of translocation.

RecQ Helicase-catalyzed DNA Unwinding Detected by Fluorescence Resonance Energy Transfer

A fluorometric assay was used to study the DNA unwinding kinetics induced by Escherichia coli RecQ helicase. This assay was based on fluorescence resonance energy transfer and carried out on stopped-flow, in which DNA unwinding was monitored by fluorescence emission enhancement of fluorescein resulting from helicase-catalyzed DNA unwinding. By this method, we determined the DNA unwinding rate of RecQ at different enzyme concentrations. We also studied the dependences of DNA unwinding magnitude and rate on magnesium ion concentration. We showed that this method could be used to determine the polarity of DNA unwinding. This assay should greatly facilitate further study of the mechanism for RecQ-catalyzed DNA unwinding.

Cold stress-induced pea DNA helicase 47 is homologous to eIF4A and inhibited by DNA-interacting ligands

Helicases are ubiquitous molecular motor proteins that play important role in maintaining the genome integrity and thus involved in plant growth and development. Here, we report the cloning of cDNA (1.64 kb) and genomic DNA (2.2 kb) of cold stress-induced pea DNA helicase 47 ( PDH47) and characterization of its encoded protein. It belongs to DEAD-box protein family and shows striking identity (93%) with tobacco eIF4A. The transcript was induced under cold (4 °C) stress. The purified PDH47 protein (47 kDa) contains ATP-/Mg 2+-dependent DNA unwinding as well as DNA-/Mg 2+-dependent ATPase activities. The ATPase activity of PDH47 is stimulated more by ssDNA as compared to dsDNA and RNA. The activities of PDH47 are inhibited by various DNA-interacting ligands such as nogalamycin, daunorubicin, ethidium bromide, mitoxantrone, actinomycin, and cisplatin with apparent K i values ranging from 0.5 to 8.0 μM. Interestingly, netropsin and distamycin inhibited the helicase but not the ATPase activity. The inhibition might be due to the intercalation of inhibitors into duplex DNA, which can impede the translocation of the PDH47. This study should help in our better understanding of cold stress signaling and mechanism of DNA unwinding in plants.

The Gly-Arg-rich C-terminal domain of pea nucleolin is a DNA helicase that catalytically translocates in the 5′- to 3′-direction

Nucleolin is a major nucleolar phosphoprotein of exponentially growing eukaryotic cells. Here we report the cloning, purification, and characterization of the C-terminal glycine/arginine-rich (GAR) domain of pea nucleolin. The purified recombinant protein (17 kDa) shows ATP-/Mg 2+-dependent DNA helicase and ssDNA-/Mg 2+-dependent ATPase activities. The enzyme unwinds DNA in the 5′- to 3′-direction, which is the first report in plant for this directional activity. It unwinds forked/non-forked DNA with equal efficiency. The anti-nucleolin antibodies immunodepleted the activities of the enzyme. The DNA interacting ligands nogalamycin, daunorubicin, actinomycin C1, and ethidium bromide were inhibitory to DNA unwinding (with K i values of 0.40, 2.21, 8.0, and 9.0 μM, respectively) and ATPase (with K i values of 0.43, 1.65, 4.6, and 7.0 μM, respectively) activities of the enzyme. This study confirms that the unwinding and ATPase activities of pea nucleolin resided in the GAR domain. This study should make important contribution to our better understanding of DNA transaction in plants, mechanism of DNA unwinding, and the mechanism by which these ligands can disturb genome integrity.

Structural basis of eukaryotic gene transcription

An RNA polymerase II promoter has been isolated in transcriptionally activated and repressed states. Topological and nuclease digestion analyses have revealed a dynamic equilibrium between nucleosome removal and reassembly upon transcriptional activation, and have further shown that nucleosomes are removed by eviction of histone octamers rather than by sliding. The promoter, once exposed, assembles with RNA polymerase II, general transcription factors, and Mediator in a ∼3 MDa transcription initiation complex. X-ray crystallography has revealed the structure of RNA polymerase II, in the act of transcription, at atomic resolution. Extension of this analysis has shown how nucleotides undergo selection, polymerization, and eventual release from the transcribing complex. X-ray and electron crystallography have led to a picture of the entire transcription initiation complex, elucidating the mechanisms of promoter recognition, DNA unwinding, abortive initiation, and promoter escape.

Effects of Temperature and ATP on the Kinetic Mechanism and Kinetic Step-size for E.coli RecBCD Helicase-catalyzed DNA Unwinding

The kinetic mechanism by which Escherichia coli RecBCD helicase unwinds duplex DNA was studied using a fluorescence stopped-flow method. Single turnover DNA unwinding experiments were performed using a series of fluorescently labeled DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. All or no DNA unwinding time courses were obtained by monitoring the changes in fluorescence resonance energy transfer between a Cy3 donor and Cy5 acceptor fluorescent pair placed on opposite sides of a nick in the duplex DNA. From these experiments one can determine the average rates of DNA unwinding as well as a kinetic step-size, defined as the average number of base-pairs unwound between two successive rate-limiting steps repeated during DNA unwinding. In order to probe how the kinetic step-size might relate to a mechanical step-size, we performed single turnover experiments as a function of [ATP] and temperature. The apparent unwinding rate constant, kUapp, decreases with decreasing [ATP], exhibiting a hyperbolic dependence on [ATP] (K1/2=176(±30)μM) and a maximum rate of kUapp=204(±4) steps s−1 (mkUapp=709(±14)bps−1) (10 mM MgCl2, 30 mM NaCl (pH 7.0), 5% (v/v) glycerol, 25.0 °C). kUapp also increases with increasing temperature (10–25 °C), with Ea=19(±1) kcal mol−1. However, the average kinetic step-size, m=3.9(±0.5) bp step−1, remains independent of [ATP] and temperature. This indicates that even though the values of the rate constants change, the same elementary kinetic step in the unwinding cycle remains rate-limiting over this range of conditions and this kinetic step remains coupled to ATP binding. The implications of the constancy of the measured kinetic step-size for the mechanism of RecBCD-catalyzed DNA unwinding are discussed.

Fluorescence Stopped-flow Studies of Single Turnover Kinetics of E.coli RecBCD Helicase-catalyzed DNA Unwinding

We have developed and optimized a stopped-flow fluorescence assay for use in studying DNA unwinding catalyzed by Escherichia coli RecBCD helicase. This assay monitors changes in fluorescence resonance energy transfer (FRET) between a pair of fluorescent probes (Cy3 donor and Cy5 acceptor) placed on opposite sides of a nick in duplex DNA. As such, this is an “all-or-none” DNA unwinding assay. Single turnover DNA unwinding experiments were performed using a series of eight fluorescent DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. The time-courses obtained by monitoring Cy3 fluorescence display a distinct lag phase that increases with increasing duplex DNA length, reflecting the transient formation of partially unwound DNA intermediates. These Cy3 FRET time-courses are identical with those obtained using a chemical quenched-flow kinetic assay developed previously. The signal from the Cy5 fluorescence probe shows additional effects that appear to specifically monitor the RecD helicase subunit. The continuous nature of this fluorescence assay enabled us to acquire more precise time-courses for many more duplex DNA lengths in a significantly reduced amount of time, compared to quenched-flow methods. Global analysis of the Cy3 and Cy5 FRET time-courses, using an n-step sequential DNA unwinding model, indicates that RecBCD unwinds duplex DNA with an average unwinding rate constant of kU=200(±40) steps s−1 (mkU=680(±12) bp s−1) and an average kinetic step size, m=3.4(±0.6) bp step−1 (5 mM ATP, 10 mM MgCl2, 30 mM NaCl, pH 7.0, 5% (v/v) glycerol, 25.0 °C), in excellent agreement with the kinetic parameters determined using quenched-flow techniques. Under these same conditions, the RecBC enzyme unwinds DNA with a very similar rate. These methods will facilitate detailed studies of the mechanisms of DNA unwinding and translocation of the RecBCD and RecBC helicases.

The DNA-unwinding mechanism of the ring helicase of bacteriophage T7.

Helicases are motor proteins that use the chemical energy of NTP hydrolysis to drive mechanical processes such as translocation and nucleic acid strand separation. Bacteriophage T7 helicase functions as a hexameric ring to drive the replication complex by separating the DNA strands during genome replication. Our studies show that T7 helicase unwinds DNA with a low processivity, and the results indicate that the low processivity is due to ring opening and helicase dissociating from the DNA during unwinding. We have measured the single-turnover kinetics of DNA unwinding and globally fit the data to a modified stepping model to obtain the unwinding parameters. The comparison of the unwinding properties of T7 helicase with its translocation properties on single-stranded (ss)DNA has provided insights into the mechanism of strand separation that is likely to be general for ring helicases. T7 helicase unwinds DNA with a rate of 15 bp/s, which is 9-fold slower than the translocation speed along ssDNA. T7 helicase is therefore primarily an ssDNA translocase that does not directly destabilize duplex DNA. We propose that T7 helicase achieves DNA unwinding by its ability to bind ssDNA because it translocates unidirectionally, excluding the complementary strand from its central channel. The results also imply that T7 helicase by itself is not an efficient helicase and most likely becomes proficient at unwinding when it is engaged in a replication complex.

Effects of Temperature and ATP on the Kinetic Mechanism and Kinetic Step-size for E. coli RecBCD Helicase-catalyzed DNA Unwinding

The kinetic mechanism by which Escherichia coli RecBCD helicase unwinds duplex DNA was studied using a fluorescence stopped-flow method. Single turnover DNA unwinding experiments were performed using a series of fluorescently labeled DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. All or no DNA unwinding time courses were obtained by monitoring the changes in fluorescence resonance energy transfer between a Cy3 donor and Cy5 acceptor fluorescent pair placed on opposite sides of a nick in the duplex DNA. From these experiments one can determine the average rates of DNA unwinding as well as a kinetic step-size, defined as the average number of base-pairs unwound between two successive rate-limiting steps repeated during DNA unwinding. In order to probe how the kinetic step-size might relate to a mechanical step-size, we performed single turnover experiments as a function of [ATP] and temperature. The apparent unwinding rate constant, kUapp, decreases with decreasing [ATP], exhibiting a hyperbolic dependence on [ATP] (K1/2=176(±30)μM) and a maximum rate of kUapp=204(±4)stepss−1(mkUapp=709(±14)bps−1) (10 mM MgCl2, 30 mM NaCl (pH 7.0), 5% (v/v) glycerol, 25.0 °C). kUapp also increases with increasing temperature (10–25 °C), with Ea=19(±1)kcalmol−1. However, the average kinetic step-size, m=3.9(±0.5)bpstep−1, remains independent of [ATP] and temperature. This indicates that even though the values of the rate constants change, the same elementary kinetic step in the unwinding cycle remains rate-limiting over this range of conditions and this kinetic step remains coupled to ATP binding. The implications of the constancy of the measured kinetic step-size for the mechanism of RecBCD-catalyzed DNA unwinding are discussed.

Fluorescence Stopped-flow Studies of Single Turnover Kinetics of E. coli RecBCD Helicase-catalyzed DNA Unwinding

We have developed and optimized a stopped-flow fluorescence assay for use in studying DNA unwinding catalyzed by Escherichia coli RecBCD helicase. This assay monitors changes in fluorescence resonance energy transfer (FRET) between a pair of fluorescent probes (Cy3 donor and Cy5 acceptor) placed on opposite sides of a nick in duplex DNA. As such, this is an “all-or-none” DNA unwinding assay. Single turnover DNA unwinding experiments were performed using a series of eight fluorescent DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. The time-courses obtained by monitoring Cy3 fluorescence display a distinct lag phase that increases with increasing duplex DNA length, reflecting the transient formation of partially unwound DNA intermediates. These Cy3 FRET time-courses are identical with those obtained using a chemical quenched-flow kinetic assay developed previously. The signal from the Cy5 fluorescence probe shows additional effects that appear to specifically monitor the RecD helicase subunit. The continuous nature of this fluorescence assay enabled us to acquire more precise time-courses for many more duplex DNA lengths in a significantly reduced amount of time, compared to quenched-flow methods. Global analysis of the Cy3 and Cy5 FRET time-courses, using an n-step sequential DNA unwinding model, indicates that RecBCD unwinds duplex DNA with an average unwinding rate constant of kU=200(±40)stepss−1 (mkU=680(±12)bps−1) and an average kinetic step size, m=3.4(±0.6)bpstep−1 (5 mM ATP, 10 mM MgCl2, 30 mM NaCl, pH 7.0, 5% (v/v) glycerol, 25.0 °C), in excellent agreement with the kinetic parameters determined using quenched-flow techniques. Under these same conditions, the RecBC enzyme unwinds DNA with a very similar rate. These methods will facilitate detailed studies of the mechanisms of DNA unwinding and translocation of the RecBCD and RecBC helicases.

Unraveling DNA helicases. Motif, structure, mechanism and function.

DNA helicases are molecular 'motor' enzymes that use the energy of NTP hydrolysis to separate transiently energetically stable duplex DNA into single strands. They are therefore essential in nearly all DNA metabolic transactions. They act as essential molecular tools for the cellular machinery. Since the discovery of the first DNA helicase in Escherichia coli in 1976, several have been isolated from both prokaryotic and eukaryotic systems. DNA helicases generally bind to ssDNA or ssDNA/dsDNA junctions and translocate mainly unidirectionally along the bound strand and disrupt the hydrogen bonds between the duplexes. Most helicases contain conserved motifs which act as an engine to drive DNA unwinding. Crystal structures have revealed an underlying common structural fold for their function. These structures suggest the role of the helicase motifs in catalytic function and offer clues as to how these proteins can translocate and unwind DNA. The genes containing helicase motifs may have evolved from a common ancestor. In this review we cover the conserved motifs, structural information, mechanism of DNA unwinding and translocation, and functional aspects of DNA helicases.

T7 박테리오파지 gp4 DNA helicase에 의한 DNA unwinding에서 step size의 반응속도론적 측정

T7박테리오파지 gp4는 dTTP 가수분해에너지를 이용하여 DNA복제시 이중 나선 DNA를 단일가닥 DNA로 풀어내는 나선효소(helicase)이다. T7 나선효소의 활성형의 4차구조는 한가운데 구멍을 지닌 육량체 고리모양이다. 단일가닥 DNA는 나선효소가 <TEX>$5'\rightarrow3'$</TEX>방향으로 이동할 때 육량체 고리의 구멍으로 빠져나간다. 이러한 DNA의 이중나선 풀어헤침을 빠른 효소반응속도 측정법을 이용하여 정량적으로 측정하였으며, 그 결과 단일가닥 DNA 산물들이 생성되기 전에 지연상태(lag phase)가 존재함을 관찰하였다. 이러한 지연상태를 나선효소에 의한 이중나선 DNA의 풀어헤침이 속도론적 단계과정(kinetic stepping)을 거친다는 모델로써 분석하였다. 예상대로 이중나선의 길이가 클수록 지연상태의 지속시간이 늘어났다. <TEX>$\tau7$</TEX> 나선효소가 이중나선 DNA를 풀어내는 과정에서 넣어준 trap DNA는 풀어내는 이중나선 DNA의 양을 변화시키지 못하여서, <TEX>$\tau7$</TEX> 나선효소가 매우 큰 공정성을 지닌 효소임을 알 수 있었다. 이러한 속도론적 data를 global fitting법을 써서 kinetic stepping 모델에 적용한 결과 매 단계(step)마다 10∼l개의 염기쌍이 풀려지고 1초당 3.7번의 step이 일어난다는 것을 알 수 있었다. DNA 풀어헤침과 dTTP가수분해의 메커니즘과 이들의 연계성은 <TEX>$4∼37^{\circ}C$</TEX>사이의 온도범위에서 영향을 받지 않았다. 이상을 종합할 때, T7나선효소의 이중나선 DNA의 풀어헤침 시 나타나는 속도론적 단계과정은 DNA복제 시 이용되는 나선효소의 내재적 속성임을 알 수 있다. T7 bacteriophage gp4 is the replicative DNA helicase that unwinds double-stranded DNA by utilizing dTTP hydrolysis energy. The quaternary structure of the active form of T7 helicase is a hexameric ring with a central channel. Single-stranded DNA passes through the central channel of the hexameric ring as the helicase translocates <TEX>$5'\rightarrow3'$</TEX> along the single-stranded DNA. The DNA unwinding was measured by rapid kinetic methods and showed a lag before the single-stranded DNA started to accumulate exponentially. This behavior was analyzed by a kinetic stepping model for the unwinding process. The observed lag phase increased as predicted by the model with increasing double-stranded DNA length. Trap DNA added in the reaction had no effect on the amplitudes of double-stranded DNA unwound, indicating that the <TEX>$\tau7$</TEX> helicase is a highly processive helicase. Global fitting of the kinetic data to the stepping model provided a kinetic step size of 10-11 bp/step with a rate of <TEX>$3.7 s^{-1}$</TEX> per step. Both the mechanism of DNA unwinding and dTTP hydrolysis and the coupling between the two are unaffected by temperature from <TEX>$4∼37^{\circ}C$</TEX>. Thus, the kinetic stepping for dsDNA unwinding is an inherent property of tile replicative DNA helicase.

DNA Unwinding Step-size of E. coli RecBCD Helicase Determined from Single Turnover Chemical Quenched-flow Kinetic Studies

The mechanism by which Escherichia coli RecBCD DNA helicase unwinds duplex DNA was examined in vitro using pre-steady-state chemical quenched-flow kinetic methods. Single turnover DNA unwinding experiments were performed by addition of ATP to RecBCD that was pre-bound to a series of DNA substrates containing duplex DNA regions ranging from 24bp to 60bp. In each case, the time-course for formation of completely unwound DNA displayed a distinct lag phase that increased with duplex length, reflecting the transient formation of partially unwound DNA intermediates during unwinding catalyzed by RecBCD. Quantitative analysis of five independent sets of DNA unwinding time courses indicates that RecBCD unwinds duplex DNA in discrete steps, with an average unwinding “step-size”, m=3.9(±1.3)bpstep−1, with an average unwinding rate of kU=196(±77)stepss−1 (mkU=790(±23)bps−1) at 25.0°C (10mM MgCl2, 30mM NaCl (pH 7.0), 5% (v/v) glycerol). However, additional steps, not linked directly to DNA unwinding are also detected. This kinetic DNA unwinding step-size is similar to that determined for the E.coli UvrD helicase, suggesting that these two SF1 superfamily helicases may share similar mechanisms of DNA unwinding.

Potent inhibition of DNA unwinding and ATPase activities of pea DNA helicase 45 by DNA-binding agents

Pea DNA helicase 45 (PDH45) is an ATP-dependent DNA unwinding enzyme, with intrinsic DNA-dependent ATPase activity [Plant J. 24 (2000) 219]. We have determined the effect of various DNA-binding agents, such as daunorubicin, ethidium bromide, ellipticine, cisplatin, nogalamycin, actinomycin C1, and camptothecin on the DNA unwinding and ATPase activities of the plant nuclear DNA helicase PDH45. The results show that all the agents except actinomycin C1, and camptothecin inhibited the helicase (apparent K i values ranging from 1.5 to 7.0 μM ) and ATPase (apparent K i values ranging from 2.5 to 11.9 μM ) activities. This is the first study to show the effect of various DNA-binding agents on the plant nuclear helicase and also first to demonstrate inhibition of any helicase by cisplatin. Another striking finding that the actinomycin C1 and ellipticine act differentially on PDH45 as compared to pea chloroplast helicase suggests that the mechanism of DNA unwinding could be different in nucleus and chloroplast. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of PDH45, resulting in both the inhibitions of unwinding activity and ATP hydrolysis. This study would be useful to obtain a better understanding of the mechanism of plant nuclear DNA helicase unwinding and the mechanism by which these agents can disturb genome integrity.

DNA Unwinding Mechanism for the Transcriptional Activation ofmomP1 Promoter by the Transactivator Protein C of Bacteriophage Mu

Transcription factor-induced conformational changes in DNA are one of the mechanisms of transcription activation. C protein of bacteriophage Mu appears to transactivate the mom gene of the phage by this mode. DNA binding by C to its site leads to torsional changes that seem to compensate for a weak momP1 promoter having a suboptimal spacing of 19 bp between the poor -35 and -10 elements. The C-mediated unwinding could realign the promoter elements for RNA polymerase recruitment to the reoriented promoter. In this study, the model has been tested by mutational analysis of the spacer region of momP1 and by assessing the strength of the mutant promoters. The response to C-mediated transactivation was dependent on the spacer length of the promoters. Mutants with 17-bp spacing between the two promoter elements showed reduced activity in the presence of the transactivator as compared with their basal level. A synthetic promoter with near consensus promoter elements and optimal 17-bp spacing was also tested to evaluate the model. The results imply a role for C-mediated unwinding in mom transcription activation.

DNA and ATP binding activities of the baculovirus DNA helicase P143.

P143 is a DNA helicase that tightly binds both double-stranded and single-stranded DNA. DNA-protein complexes rapidly dissociated in the presence of ATP and Mg(2+). This finding suggests that ATP hydrolysis causes a conformational change in P143 which decreases affinity for DNA. This supports the model of an inchworm mechanism of DNA unwinding.

Analysis of sigma(54)-dependent genes in Enterococcus faecalis: a mannose PTS permease (EII(Man)) is involved in sensitivity to a bacteriocin, mesentericin Y105.

The sigma(54) RNA polymerase subunit has a prominent role in susceptibility of Listeria monocytogenes and Enterococcus faecalis to mesentericin Y105, a class IIa bacteriocin. Consequently, sigma(54)-dependent genes as well as specific activators also required for expression of these genes were sought. Five putative sigma(54)-associated activators were detected in the genome of E. faecalis V583, and all but one could activate the transcription of permease genes belonging to sugar phosphotransferase systems (PTSs). Interestingly, these activators display a helicase signature not yet reported in this activator family, which could explain the ATP-dependent mechanism of DNA unwinding preceding the start of transcription. To find which activator is linked to susceptibility of E. faecalis to mesentericin Y105, their respective genes were subsequently interrupted. Among them, only mptR gene interruption led to a resistance phenotype. Immediately downstream from mptR, a putative sigma(54)-dependent operon was found to encode a mannose PTS permease, namely EII(t)(Man). Moreover, in liquid culture, glucose and mannose induced the sensitivity of E. faecalis to mesentericin Y105. Since sugars have previously been reported to induce PTS permease expression, it appears that EII(t)(Man) expression, presumably induced in the presence of glucose and mannose, leads to an enhanced sensitivity of E. faecalis to the bacteriocin. Additional information was gained from knockouts within the permease operon. Interruption of the distal mptD gene, which encodes the IID subunit of EII(t)(Man), strikingly led to resistance to mesentericin Y105. Moreover, MptD appears to be a peculiar membrane subunit, bearing an additional domain compared to most known IID subunits. According to these results, EII(t)(Man) is clearly involved in susceptibility to mesentericin Y105 and could even be its receptor at the E. faecalis surface. Finally, it is hypothesized that MptD could be responsible for the targeting specificity, via an interaction between its additional domain and mesentericin Y105.

Crystal structure of the hexameric replicative helicase RepA of plasmid RSF1010

Unwinding of double-stranded DNA into single-stranded intermediates required for various fundamental life processes is catalyzed by helicases, a family of mono-, di- or hexameric motor proteins fueled by nucleoside triphosphate hydrolysis. The three-dimensional crystal structure of the hexameric helicase RepA encoded by plasmid RSF1010 has been determined by X-ray diffraction at 2.4 Å resolution. The hexamer shows an annular structure with 6-fold rotational symmetry and a ∼17 Å wide central hole, suggesting that single-stranded DNA may be threaded during unwinding. Homologs of all five conserved sequence motifs of the DnaB-like helicase family are found in RepA, and the topography of the monomer resembles RecA and the helicase domain of the bacteriophage T7 gp4 protein. In a modeled complex, ATP molecules are located at the subunit interfaces and clearly define adenine-binding and ATPase catalytic sites formed by amino acid residues located on adjacent monomers; most remarkable is the “arginine finger” Arg207 contributing to the active site in the adjacent monomer. This arrangement of active-site residues suggests cooperativity between monomers in ATP hydrolysis and helicase activity of RepA. The mechanism of DNA unwinding remains elusive, as RepA is 6-fold symmetric, contrasting the recently published asymmetric structure of the bacteriophage T7 gp4 helicase domain.

Characterisation of the catalytically active form of RecG helicase.

Replication of DNA is fraught with difficulty and chromosomes contain many lesions which may block movement of the replicative machinery. However, several mechanisms to overcome such problems are beginning to emerge from studies with Escherichia coli. An important enzyme in one or more of these mechanisms is the RecG helicase, which may target stalled replication forks to generate a four-stranded (Holliday) junction, thus facilitating repair and/or bypass of the original lesion. To begin to understand how RecG might catalyse regression of fork structures, we have analysed what the catalytically active form of the enzyme may be. We have found that RecG exists as a monomer in solution as measured by gel filtration but when bound to junction DNA the enzyme forms two distinct protein-DNA complexes that contain one and two protein molecules. However, mutant inhibition studies failed to provide any evidence that RecG acts as a multimer in vitro. Additionally, there was no evidence for cooperativity in the junction DNA-stimulated hydrolysis of ATP. These data suggest that RecG functions as a monomer to unwind junction DNA, which supports an 'inchworm' rather than an 'active rolling' mechanism of DNA unwinding. The observed in vivo inhibition of wild-type RecG by mutant forms of the enzyme was attributed to occlusion of the DNA target and correlates with the very low abundance of replication forks within an E.COLI: cell, even during rapid growth.