Biomedical subjects

D Bastia

Publications and source records attributed to D Bastia.

At least 37 records · Page 2Linked to original sources

PubMed1995

The contrahelicase activities of the replication terminator proteins of Escherichia coli and Bacillus subtilis are helicase-specific and impede both helicase translocation and authentic DNA unwinding.

Replication forks are arrested at sequence-specific replication termini primarily, perhaps exclusively, by polar arrest of helicase-catalyzed DNA unwinding by the terminator protein. The mechanism of this arrest is of considerable interest. This paper presents experimental evidence in support of four major points pertaining to termination of DNA replication. First, the replication terminator proteins of both Escherichia coli and Bacillus subtilis are helicase-specific contrahelicases, i.e. the proteins specifically impede the activities of helicases that are involved in symmetric DNA replication but not of those involved in conjugative DNA transfer and rolling circle replication. Second, the terminator protein (Ter) of E. coli blocks not only helicase translocation but also authentic DNA unwinding. Third, the replication terminator protein of Gram-positive B. subtilis is a polar contrahelicase of the primosomal helicase PriA of Gram-negative E. coli. Finally, the blockage of PriA-catalyzed DNA unwinding was abrogated by the passage of an RNA transcript through the replication terminator protein-terminus complex. These results are significant because of their relevance to the mechanistic aspects of replication termination.

Bacillus subtilis↗

PubMed1995

Crystal structure of the replication terminator protein from B. subtilis at 2.6 A.

The crystal structure of the replication terminator protein (RTP) of B. subtilis has been determined at 2.6 A resolution. As previously suggested by both biochemical and biophysical studies, the molecule exists as a symmetric dimer and is in the alpha + beta protein-folding class. The protein has several uncommon features, including an antiparallel coiled-coil, which serves as the dimerization domain, and both an alpha-helix and a beta-ribbon suitably positioned to interact with the major and minor grooves of B-DNA. A site has been identified on the surface of RTP that is biochemically and positionally suitable for interaction with the replication-specific helicase. Other features of the structure are consistent with the polar contrahelicase mechanism of the protein. A model of the interaction between RTP and its cognate DNA is presented.

Amino Acid Sequence↗

PubMed1995

Termination of DNA replication in vitro: requirement for stereospecific interaction between two dimers of the replication terminator protein of Bacillus subtilis and with the terminator site to elicit polar contrahelicase and fork impedance.

The termination of DNA replication at a sequence-specific replication terminus in Bacillus subtilis is catalyzed by a dimeric replication terminator protein (RTP) of subunit mol. wt 14,500. RTP has become an attractive protein with which to study the molecular mechanism of termination because its crystal structure has now been solved and the previous lack of an in vitro replication system has been largely overcome by our discovery that the protein terminates replication in vivo and in vitro in the well-studied Gram-negative Escherichia coli system. We have exploited the surrogate in vitro system to show that RTP acts as a polar contrahelicase to DnaB helicase of E. coli only when two RTP dimers are bound co-operatively to overlapping core and auxiliary sequences comprising the terminus. A core sequence by itself binds one dimer of RTP, but elicits no contrahelicase activity. Binding of two RTP dimers to a tandem head-to-tail core repeat also elicits no contrahelicase activity, thus suggesting that a specific stereochemical interaction between two RTP dimers and with the terminator site is essential for termination. RTP blocks unwinding of DNA substrates containing heteroduplex regions that include the terminus and are in the size range of approximately 50 to > 1000 bp in length. Thus, the protein blocks authentic helicase-catalyzed unwinding rather than just the translocation of the helicase on DNA.

Bacillus subtilis↗

PubMed1994

The replication terminator protein of the gram-positive bacterium Bacillus subtilis functions as a polar contrahelicase in gram-negative Escherichia coli.

The replication terminator protein (RTP) of Bacillus subtilis is a dimer with a monomeric molecular mass of 14.5 kDa. The protein terminates DNA replication at a specific binding site. Although the protein has been crystallized and its crystal structure has been solved, the lack of an in vitro replication system in B. subtilis has been a serious impediment to the analysis of the mechanism of action of this protein. We have discovered that the protein is functional in the Gram-negative bacterium Escherichia coli in vivo and in vitro. RTP blocked replication forks initiated from a ColE1 replication origin at the cognate DNA-binding site (BS3) in a polar mode. The protein did not block rolling circle replication initiated from the pT181 origin in cell extracts of Staphylococcus aureus. RTP antagonized the helicase activity of DnaB but not that of helicase II of E. coli. Thus, RTP functioned as a polar contrahelicase blocking a helicase that participates in symmetric DNA replication but it did not impede rolling circle replication nor the action of a helicase involved in DNA repair.

Adenosine Triphosphatases↗

PubMed1994

Multiple pathways of copy control of gamma replicon of R6K: mechanisms both dependent on and independent of cooperativity of interaction of tau protein with DNA affect the copy number.

The ability of a replication initiator protein to promote intermolecular pairing of two replication origins resulting in the turning off of the origin pair has been called handcuffing. We have endeavored to test the validity of the handcuffing model by isolating two mutant forms of the tau initiator protein of R6K that elicit high copy number phenotype. We have discovered that one mutant called tau 113 yielded a 3.6-fold increase in copy number of a gamma replicon with a concomitant impairment of its ability to loop DNA and to pair binding sites (iterons) in comparison with normal tau, thus supporting the handcuffing model. A second mutant called tau 108, on the other hand, elicited a 3-fold increase in copy number without showing any measurable loss in its ability to loop and pair gamma iterons. Both mutant forms of the wild-type tau protein showed no detectable differences in their affinity of binding to the gamma iterons. Thus, the phenotype of tau 108 is consistent with the proposition that copy number control involves macromolecular interactions other than cooperativity at a distance of tau or interaction of tau with the primary binding sites at gamma. Taken together, the results are consistent with the notion that tau-mediated handcuffing is a mechanism, but not the only mechanism, of copy control in R6K. Interaction of tau with host proteins is likely to provide additional facets of the copy control mechanism.

Amino Acid Sequence↗

PubMed1993

A 27 kd protein of E. coli promotes antitermination of replication in vitro at a sequence-specific replication terminus.

We have discovered a 27 kd protein of E. coli that binds to a terminator site (tau)-terminator protein (ter) complex and abrogates the replication fork-arresting activity of ter protein in vitro. The 27 kd protein also neutralizes the contrahelicase activity of ter protein, allowing dnaB helicase to unwind DNA past a tau-ter complex. The stimulatory activity of low levels of ter protein on helicase II is also abolished by the 27 kd protein. The binding of the 27 kd protein to a tau-ter complex does not appear to dissociate the ter protein from the DNA. Although the in vivo function of the 27 kd protein is unknown at this time, it has the major attributes of a novel replication antiterminator in vitro.

Bacterial Proteins↗

PubMed1992

Crystallization and preliminary structural analysis of the replication terminator protein of Bacillus subtilis.

The replication terminator protein (RTP) is a dimeric molecule that binds specific sequences within the replication terminus of the Bacillus subtilis chromosome and prevents the passage of replication forks. The gene for RTP has been expressed in Escherichia coli, and the protein has been purified in amounts sufficient for structural studies by nuclear magnetic resonance (NMR) and x-ray crystallography. One-dimensional NMR experiments show that the protein has a well-folded compact tertiary structure, as well as a high alpha-helical content. Circular dichroism (CD) studies confirm this finding and show that approximately 32% of the protein is alpha-helical. The terminator protein has been crystallized as monoclinic plates that diffract to better than 2.5 A and are suitable for high resolution structural analysis. Precession photographs show the space group to be C2 with unit cell dimensions a = 77 A, b = 53 A, c = 70 A, and beta = 90 degrees, and two molecules occupy the asymmetric unit. With a view to producing crystals of an RTP.DNA complex, gel-shift assays were performed to establish the shortest sequence of DNA that is required for tight binding to RTP. These clearly show that two turns of DNA are required, centered on an 8-base pair consensus sequence, to elicit relatively stable binding.

Autoradiography↗

PubMed1992

Structural and functional analysis of a replication enhancer: separation of the enhancer activity from origin function by mutational dissection of the replication origin gamma of plasmid R6K.

The plasmid R6K possesses three distinct origins of replication: alpha, beta, and gamma. The replication origin gamma of plasmid R6K performs a dual function: (i) as an origin itself and (ii) as an enhancer element required in cis for the activation at a distance of the other two replication origins alpha and beta. We have dissected the gamma origin/enhancer by site-directed mutagenesis and have reached the following conclusions. The origin function can be specifically inactivated without impairing the enhancer function by insertion and/or deletion mutations near the opposite ends of the origin gamma sequence. One such mutation deleted sequences that included the left DnaA site I. The second mutation involved insertion of linker sequences that resulted in a spatial alteration between the right DnaA site II and the VIIth pi binding iteron (tandemly repeated binding sites). Other mutations that either partly or completely deleted the A+T-rich sequence adjacent to, but not including, the pi binding iterons also abrogated enhancer and origin function and suggested that pi binding sites were necessary but not sufficient for enhancer activity. Finally, the functional analysis of a set of mutants of the gamma origin/enhancer suggested that a continuous stretch of 300 base pairs is necessary for origin gamma function and that the sequences that included the binding sites for pi, DnaA, and integration host factor proteins are required in the correct stereochemical alignment to impart origin activity.

Bacterial Proteins↗

PubMed1992

Activation of distant replication origins in vivo by DNA looping as revealed by a novel mutant form of an initiator protein defective in cooperativity at a distance.

We have isolated mutants of the pi initiator protein of the plasmid R6K that are defective in DNA looping in vitro but retain their normal DNA binding affinity for the primary binding sites (iterons) at the gamma origin/enhancer. One such looping defective mutant called R6 was determined to be a proline to leucine change at position 46 near the N terminus of the pi protein. Using a set of genetic assays that discriminate between the activation of the gamma origin/enhancer from those of the distantly located alpha and beta origins, we show that the looping defective initiator protein fails to activate the alpha and beta origins but derepresses initiation from the normally silent gamma origin in vivo. The results conclusively prove that DNA looping is required to activate distant replication origins located at distances of up to 3 kb from the replication enhancer.

Autoradiography↗

PubMed1992

Activation in vivo of the minimal replication origin beta of plasmid R6K requires a small target sequence essential for DNA looping.

The plasmid R6K contains three distinct origins of replication: alpha, beta, and gamma. The gamma sequence is essential in cis and acts as an enhancer that activates the distant alpha and beta origins. R6K therefore represents a favorable procaryotic model system with which to unravel the biochemical mechanisms underlying selective origin activation, particularly activation involving distant sites on the same chromosome. We have discovered that plasmids containing the origins alpha and gamma required the Escherichia coli DnaA initiator protein in addition to the R6K-encoded initiator protein, Pi, and other host replisomal proteins for their maintenance in vivo. Plasmids initiating replication from origin beta required only the Pi initiator protein and other host replisomal proteins. We have exploited the differential requirement for the DnaA protein by origins gamma and beta to selectively study and localize the minimal origin beta sequences by deletion analysis as one test of a looping model of origin activation. A 64-bp region spanning the extreme -COOH terminal coding sequence of the Pi protein was found to be essential for replication in vivo in the absence of DnaA protein, consistent with the approximate physical location of the beta origin. Replication emanating from origin beta could be abolished in vivo by deletion of the 9-bp target site for Pi protein-mediated DNA looping between the gamma origin/enhancer and the distant beta origin. Electron microscopy of nascent replication intermediates generated in vivo directly confirmed our genetic localization of the beta origin. Our results strongly suggest that activation of the beta origin by a distant replication enhancer element requires a small target sequence essential for initiator protein-mediated DNA looping.

Amino Acid Sequence↗

PubMed1991

Conformational changes induced by integration host factor at origin gamma of R6K and copy number control.

We have investigated the role of integration host factor (IHF) in the replication of plasmid R6K by studying the maintainance of the plasmid in a strain of Escherichia coli that lacks both subunits of IHF and in an isogenic wild type strain and found that all three origins, alpha, beta, and gamma, were functional in the absence of IHF; however, loss of IHF reduced the copy number of those replicons initiating solely from ori gamma by 5-fold. Concomitant loss of direct repeats within the origin that bind the R6K replication initiator protein, Pi, resulted in a further reduction in copy number. Using gel mobility shift analysis, we showed that IHF bound specifically only to one site within the A/T rich region of the minimal origin adjacent to the Pi binding sites. The origin region possessed no intrinsic DNA curvature although IHF induced a strong bend upon binding. Combination footprinting with different orders of addition of Pi and IHF suggested that there was no cooperativity between the two proteins with regard to DNA binding. Hydroxyl-radical footprinting revealed hypersensitive asymmetric periodic cleavage sites within the origin region in the presence of IHF that extended over 200 base pairs and a localized perturbation of cleavage chemistry. The presence of periodic cleavages was dependent upon the presence of the wild type R6K origin sequence and was not observed when the IHF binding site was positioned adjacent to a heterologous sequence. We observed that the conformational changes induced by IHF upon binding to the R6K origin were negatively correlated with the observed decrease in copy number, and therefore, origin conformation altered by protein-DNA interaction may play an important role in the regulation of replication initiation.

Autoradiography↗

PubMed1991

Replication of plasmid R6K origin gamma in vitro. Dependence on dual initiator proteins and inhibition by transcription.

We have developed a more efficient in vitro replication system for the plasmid R6K with the objective of dissecting the mechanism of activation of replication origins at a distance. Using this in vitro system we have shown that the activation of replication origin gamma of R6K is absolutely dependent on two exogenously added initiator proteins: namely the host-encoded DnaA and the plasmid-encoded Pi proteins. Replication was inhibited by novobiocin, suggesting a requirement for DNA gyrase. Surprisingly, rifampicin stimulated in vitro replication significantly, and this stimulation was manifested in the quantitative enhancement of replication without any noticeable qualitative change in the reaction products. This result suggests that transcription at or near the gamma origin keeps it repressed. Replication intermediates that were allowed to accumulate by dideoxynucleoside triphosphate incorporation were analyzed both by restriction enzyme digestion and by electron microscopy, and both sets of analyses revealed initiation from the gamma origin resulting in theta-type replication intermediates. Further development of this system should help us to understand how DNA-protein interaction at the gamma origin/enhancer activates the distal origins alpha and beta.

Autoradiography↗

PubMed1991

Replication terminator protein of Escherichia coli is a transcriptional repressor of its own synthesis.

We have investigated the regulation of synthesis of the replication terminator protein (Ter) of Escherichia coli and have discovered that the protein is a repressor of its own synthesis at the transcriptional step. Since the synthesis of Ter protein was observed to be down-regulated in vivo, these results are consistent with autoregulation as one control mechanism of Ter protein within the cell. Analysis of the tus gene that encodes the Ter protein revealed that transcription was initiated from a single promoter located within the upstream nontranscribed sequence. In vitro footprinting experiments have revealed that Ter protein prevented binding of RNA polymerase to the promoter sequence when both proteins were incubated with promoter DNA. However, once bound to the promoter, RNA polymerase could not be displaced by Ter protein. Conversely, prebound Ter protein could not be dislodged from its binding site at the promoter when challenged with RNA polymerase. Therefore, Ter protein can serve as a transcriptional repressor of its own synthesis by preventing RNA polymerase from binding to the tus promoter when both proteins are present in the cell milieu.

Bacterial Proteins↗

PubMed1991

Escherichia coli replication terminator protein impedes simian virus 40 (SV40) DNA replication fork movement and SV40 large tumor antigen helicase activity in vitro at a prokaryotic terminus sequence.

We have discovered that the Escherichia coli terminator protein (Ter) impedes replication fork movement, initiated in vitro from the simian virus 40 replication origin by the large tumor antigen (TAg), at the terminator site (tau R) of the prokaryotic plasmid R6K preferentially when tau R is present in one orientation with respect to the origin. We also have discovered that Ter impedes helicase activity of TAg at the tau R site, when tau R is in this same orientation. In contrast with Ter, a mutant EcoRI protein (EcoRIgln111) that binds with high affinity to but does not cleave at EcoRI recognition sequences impedes both simian virus 40 fork movement and the helicase activity of TAg in an EcoRI-site-orientation-independent manner. These results suggest that a feature common to both TAg and prokaryotic helicases may recognize the Ter-tau R complex resulting in a polarized pause in fork propagation and DNA unwinding. In contrast, the effect of EcoRIgln111-DNA complex on these reactions may be based on steric hindrance.

Antigens, Polyomavirus Transforming↗

PubMed1991

DNA-protein interaction at the replication termini of plasmid R6K.

Understanding the molecular mechanism of specific and polarized termination of DNA replication at a sequence-specific replication terminus requires detailed analyses of the interaction of terminator protein (ter) with specific DNA sequences (tau), constituting the replication terminus. Such analyses should provide the structural basis of the functional polarity of replication inhibition observed in vivo and in vitro at tau sites. With this objective in mind, we have purified the replication terminator protein of Escherichia coli to homogeneity and have analyzed the interaction of the protein with the replication termini of R6K, using chemical probes and by site-directed mutagenesis. The results show that one monomer of ter protein binds to a single tau site with an equilibrium dissociation constant of 5 x 10(-9) moles/liter. Furthermore, a combination of alkylation interference and protection, hydroxyradical footprinting, and site-directed mutagenesis has revealed the phosphate groups and base residues of the tau core sequence that make contacts with ter protein and those residues that are important for both DNA-protein interaction and for termination of replication in vivo. The overall picture that emerges from these analyses reveals that ter forms an asymmetric complex with a tau sequence. Thus, the asymmetric ter-tau complex provides a structural basis for the functional polarity of the arrest of a moving replication fork at a tau site.

Autoradiography↗

PubMed1991

Cooperativity at a distance promoted by the combined action of two replication initiator proteins and a DNA bending protein at the replication origin of pSC101.

We have investigated the interaction of the host-encoded DNA bending protein IHF, the host-encoded initiator DnaA, and the plasmid-encoded initiator RepA with the replication origin of pSC101. We have discovered that DNA bending induced by IHF in vitro promoted the interaction of DnaA protein with two physically separated binding sites called dnaAs and dnaAw. This cooperative interaction at a distance, most probably, caused looping out of the ihf site. We have also discovered that RepA protein binding to its cognate sites promoted enhanced binding of DnaA protein to the physically distant dnaAs site, probably also by DNA looping. The addition of RepA to a binding reaction containing IHF and DnaA further enhanced the binding of DnaA protein to the dnaAs site. Thus, the three DNA-binding proteins interacted with the origin, generating a higher order structure in vitro. On the basis of the results of the known requirement of all three proteins for replication initiation, we have proposed a model for the structure of a preinitiation complex at the replication origin.

Bacterial Proteins↗

PubMed1990

The DNA-binding domain of HPV-16 E2 protein interaction with the viral enhancer: protein-induced DNA bending and role of the nonconserved core sequence in binding site affinity.

We expressed the carboxy-terminal portion of the E2 open reading frame (ORF)-encoded protein of human papillomavirus type 16 (HPV-16) and purified it to near homogeneity. Using DNase I footprinting techniques, we show that like the homologous protein from bovine papillomavirus type 1 (BPV-1), HPV-18, HPV-11, it binds DNA at the enhancer consensus motif ACCN6GGT. Base and phosphate backbone contact points were determined using methylation protection and interference and ethylation interference assays. This HPV-16 E2 DNA-binding domain protein contacts the site at the outermost conserved GG residues which is similar to the interaction of the BPV-1 E2 system. However, there are many fewer phosphate backbone contacts. Using gel retardation assays, the HPV-16 E2 protein interaction with the consensus motif was characterized further based on the specific sequence of the noncontacted, nonconserved internal bases. Affinity of this E2 protein for the consensus site increased dramatically with an A.T-rich core sequence. Like the homologous BPV-1 protein, HPV-16 E2 protein induces DNA bending at its binding site. Furthermore, examination of the DNA region containing a single consensus motif far upstream from the major promoter, P97, revealed naturally bent DNA that was further bent upon interaction with the HPV-16 E2 protein.

Alkylation↗

PubMed1990

Sequence-specific and polarized replication termination in vitro: complementation of extracts of tus- Escherichia coli by purified Ter protein and analysis of termination intermediates.

We have developed an in vitro replication system in which purified replication termination protein (Ter) elicits specific and polarized termination of DNA replication at terminator sites (tau) in a cell extract of tus- Escherichia coli that does not encode Ter protein. Using this system and two-dimensional agarose gel electrophoresis, we have identified intermediates with stalled replication forks. The replication bubbles contain both arrested leading strands and lagging strands that were initiated at the ColE1 origin of replication and that had progressed unidirectionally until arrested at tau. To dissect the system further, we have analyzed the kinetics of the formation of the termination intermediates and have discovered that the earliest termination intermediate had properties consistent with an arrested D loop. The D loop contained an arrested leading strand. Thus, in this test system, there appears to be a transient uncoupling of leading- and lagging-strand synthesis during termination of replication at tau sites.

DNA Replication↗