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H Buc

Publications and source records attributed to H Buc.

At least 19 recordsLinked to original sources
PubMed2001

The HIV-1 repeated sequence R as a robust hot-spot for copy-choice recombination.

Template switching during reverse transcription is crucial for retroviral replication. While strand transfer on the terminal repeated sequence R is essential to achieve reverse transcription, template switching from internal regions of the genome (copy choice) leads to genetic recombination. We have developed an experimental system to study copy-choice recombination in vitro along the HIV-1 genome. We identify here several genomic regions, including the R sequence, where copy choice occurred at high rates. The frequency of copy choice occurring in a given region of template was strongly influenced by the surrounding sequences, an observation that suggests a pivotal role of the folding of template RNA in the process. The sequence R, instead, constituted an exception to this rule since it was a strong hot-spot for copy choice in the different sequence contexts tested. We suggest therefore that the structure of this region has been optimised during viral evolution to ensure efficient template switching independently from the sequences that might surround it.

Genome, Viral↗
PubMed2001

DNA synthesis by HIV-1 reverse transcriptase at the central termination site: a kinetic study.

Human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase (RT) terminates plus-strand DNA synthesis at the center of the HIV-1 genome, a process important for HIV-1 infectivity. The central termination sequence contains two termination sites (Ter1 and Ter2) located at the 3'-end of A(n)T(m) motifs, and the narrowing of the DNA minor groove generated by these motifs is responsible for termination. Kinetic data associated with the binding of RT and its ability to elongate in vitro various DNA duplexes and triplexes surrounding the Ter2 terminator were analyzed using a simple kinetic scheme. At Ter2, RT still displays a reasonable affinity for the corresponding DNA, but the binding of the next nucleotide and above all its incorporation rate are markedly hampered. Features affecting the width of the minor groove act directly at this last step. The constraint exerted against elongation by the A(n)T(m) tract persists at two positions downstream of the terminator.

Base Sequence↗
PubMed2001

Structures of complexes formed by HIV-1 reverse transcriptase at a termination site of DNA synthesis.

This study presents structural parameters associated with termination of human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase (RT) at Ter2, the major termination site located in the center of the HIV-1 genome. DNA footprinting studies of various elongation complexes formed by RT around wild type and mutant Ter2 sites have revealed two major structural transformations of these complexes when the enzyme gets closer to Ter2. First, the interactions between RT and the DNA duplex are less extended, although the global affinity of the enzyme for this duplex is only decreased by 2-fold. Second, there is an atypical positioning of the RT RNase H domain on the DNA duplex. We interpret our data as indicating that the A(n)T(m) motif located upstream of Ter2 prevents a classical positioning of the enzyme on the double-stranded part of the DNA duplex at some precise positions of elongation downstream of this motif. Instead, novel species of binary and/or ternary complexes, characterized by atypical footprints, are formed. The new rate-limiting step of the reaction, characterized in the preceding paper (Lavigne, M., Polomack, L., and Buc, H. (2001) J. Biol. Chem. 276, 31429-31438), would be a transition leading from these new species to a catalytically competent ternary complex.

Base Sequence↗
PubMed2001

The Escherichia coli RNA polymerase.anti-sigma 70 AsiA complex utilizes alpha-carboxyl-terminal domain upstream promoter contacts to transcribe from a -10/-35 promoter.

During infection of Escherichia coli, the phage T4 early protein AsiA inhibits open complex formation by the RNA polymerase holoenzyme Efinal sigma(70) at -10/-35 bacterial promoters through binding to region 4.2 of the final sigma(70) subunit. We used the -10/-35 lacUV5 promoter to study the properties of the Efinal sigma(70). AsiA complex in the presence of the glutamate anion. Under these experimental conditions, inhibition by AsiA was significantly decreased. KMnO(4) probing showed that the observed residual transcriptional activity was due to the slow transformation of the ternary complex Efinal sigma(70). AsiA.lacUV5 into an open complex. In agreement with this observation, affinity of the enzyme for the promoter was 10-fold lower in the ternary complex than in the binary complex Efinal sigma(70).lacUV5. A tau plot analysis of abortive transcription reactions showed that AsiA binding to Efinal sigma(70) resulted in a 120-fold decrease in the second-order on-rate constant of the reaction of Efinal sigma(70) with lacUV5 and a 55-fold decrease in the rate constant of the isomerization step leading to the open complex. This ternary complex still responded to activation by the cAMP.catabolite activator protein complex. We show that compensatory Efinal sigma(70)/promoter upstream contacts involving the C-terminal domains of alpha subunits in Efinal sigma(70) become essential for the binding of Efinal sigma(70). AsiA to the lacUV5 promoter.

Base Sequence↗
PubMed2001

Retroviral recombination: what drives the switch?

The high rate of recombination in retroviruses is due to the frequent template switching that occurs during reverse transcription. Although the mechanism that leads to this switch is still a matter of debate, there is increasing evidence that specific RNA structures are involved. And the implications might go beyond retroviral genetic variability.

Recombination, Genetic↗
PubMed2001

A molecular mechanism for the repression of transcription by the H-NS protein.

The H-NS protein is a major component of the bacterial nucleoid and plays a crucial role in the global gene regulation of enteric bacteria. Although H-NS does not exhibit a high DNA sequence specificity, a number of H-NS-responsive promoters have been shown to contain regions of intrinsic DNA curvature located either upstream or downstream of the transcription start point. We have studied H-NS binding to DNA and in vitro transcriptional regulation by H-NS at several synthetic promoters with or without curved sequences inserted upstream of the Pribnow box. We show how such inserts determine the final organization of H-NS-containing nucleoprotein complexes and how this affects transcription. We refine a two-step mechanism for the constitution of H-NS assemblies that are efficient in regulation.

Bacterial Proteins↗
PubMed2001

Mechanisms of retroviral recombination.

Recombination is a major source of genetic variability in retroviruses. Each viral particle contains two single-stranded genomic RNAs. Recombination mostly results from a switch in template between these two RNAs during reverse transcription. Here we emphasize the main mechanisms underlying recombination that are emerging from recent advances in biochemical and cell culture techniques. Increasing evidence supporting the involvement of RNA secondary structures now complements the predominant role classically attributed to enzyme pausing during reverse transcription. Finally, the implications of recombination on the dynamics of emergence of genomic aberrations in retroviruses are discussed.

DNA Replication↗
PubMed2000

Copy-choice recombination by reverse transcriptases: reshuffling of genetic markers mediated by RNA chaperones.

Copy-choice recombination efficiently reshuffles genetic markers in retroviruses. In vivo, the folding of the genomic RNA is controlled by the nucleocapsid protein (NC). We show that binding of NC onto the acceptor RNA molecule is sufficient to enhance recombination, providing evidence for a mechanism where the structure of the acceptor template determines the template switch. NC as well as another RNA chaperone (StpA) converts recombination into a widespread process no longer restricted to rare hot spots, an effect maximized when both the NC and the reverse transcriptase come from HIV-1. These data suggest that RNA chaperones confer a higher genetic flexibility to retroviruses.

Genetic Markers↗
PubMed1999

Simultaneous determination of pyrimidine or purine deoxyribonucleoside triphosphates using a polymerase assay.

In this paper, we describe an improved enzymatic assay for the determination of deoxyribonucleoside triphosphates (dNTPs). This is based on the elongation of 32P 5'-end-labeled oligonucleotide primers annealed to complementary oligonucleotide templates. Incorporation within the primer/template (p/t) was catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I under conditions where the concentration of the dNTP to be analyzed is limiting. Using a combination of two different sized p/t pairs, dCTP and dTTP (or dATP and dGTP) were assayed together. Since the elongated products were clearly separated after electrophoresis on a denaturing 10% polyacrylamide gel, the two dNTPs could be quantified in a single lane. This method allows for the first time the simultaneous determination of two pyrimidine or two purine deoxyribonucleoside triphosphates. Consequently, a large number of biological samples can be tested in a single experiment. The high sensitivity of this method enables the quantification of low concentrations of dNTPs, such as those found in resting nondividing cells. Furthermore, this new protocol is well suited for the determination of dNTPs in cells treated with the antiretroviral ddI, since the Klenow fragment has a low affinity for ddATP, the active form of ddI.

Anti-HIV Agents↗
PubMed1999

Studies of contacts between T7 RNA polymerase and its promoter reveal features in common with multisubunit RNA polymerases.

We have used UV-laser mediated cross-linking, DNase I footprinting and KMnO4 reactivity to probe the interaction between T7 RNA polymerase (RNAP) and a consensus promoter during the early stages of transcription. In a binary complex formed in the absence of substrate on a supercoiled plasmid, direct contacts were observed on the template (T) strand at positions -17, -5, and +3 and on the nontemplate (NT) strand at position -8. These contacts lie within the DNase I cleavage footprint from positions -21 to +11 on the T strand and from positions -17 to +16 on the NT strand and straddle sites of enhanced reactivity of thymines to KMnO4 at position -3 on the T strand and position -2 on the NT strand. Use of supercoiled plasmid templates has allowed the mapping of contacts in the initiation region of the promoter in the binary complex for the first time. Upon addition of GTP, T7 RNAP enters a reiterative mode of synthesis, producing a ladder of poly(G) products. Under these conditions the downstream contact on the T strand switched from position +3 to +4 and +5 while the contact at position -17 was maintained. Under conditions in which the synthesis of transcription products is limited to 6-7 nucleotides, only the contact at position -17 on the T strand was preserved. A comparison of these results with the interaction of Escherichia coli RNA polymerase at the lac promoter reveals strong similarities in the manner in which these polymerases recognize their promoters.

Bacteriophage T7↗
PubMed1999

Recombination during reverse transcription: an evaluation of the role of the nucleocapsid protein.

The human immuno deficiency virus type 1 nucleocapsid protein 7 (HIV-1 NCp7) is a major component of the reverse transcription complex. Its effect on reverse transcription and homologous recombination has been studied in vitro under strictly identical experimental conditions. For high enzyme concentrations, NCp7 did not stimulate DNA synthesis. The time-course for completion of reverse transcription as well as the processivity and the pattern of pausing were similar in the presence or absence of NCp7. However, the addition of NCp7 significantly affected the yield of the reaction, a decrease exacerbated as the length of the copied RNA increased. We attribute this phenomenon to a destabilization of the RNA/DNA duplex at intermediate stages of reverse transcription.In contrast, NCp7 enhanced homologous recombination during synthesis mediated by HIV-1 RT (reverse transcriptase), as it did for Moloney murine leukemia virus RT. On naked RNA the process of recombination was dependent on the concentration of RT, suggesting that binding of RT to an intermediate of strand transfer was the limiting step. This dependence was relieved in the presence of NCp7. This effect does not imply a direct interaction between RT and NCp7, since similar results were obtained when NCp7 was substituted by the bacterial RNA chaperon StpA. The dominant effect of NCp7 is therefore most probably exerted at the level of condensation of the RNA templates, leading to the formation of productive interactions between the nascent DNA and the acceptor template.

Capsid↗
PubMed1999

Compression of the DNA minor groove is responsible for termination of DNA synthesis by HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase (RT) generally terminates plus strand DNA synthesis at the centre of the viral genome. The central termination sequence (CTS) contains two termination sites which are located at the 3' end of AnTm motifs. These motifs generate a global curvature of the DNA helix which correlates with termination of DNA synthesis. Here, we have characterized HIV-1 RT termination sites on different DNA sequences. Again, they are located at the 3' end of A-tracts. Using hydroxyl radicals as a probe of the width of the DNA helix, we have shown that RT termination sites are always located a few base-pairs downstream of a compressed minor groove. Mutations which relieve these compressions also abolish the termination events. The replacement of the adenine tracts by 2,6-diaminopurine tracts has a similar effect. Moreover, no termination site is observed on DNA sequences containing phased GC-tracts which curve the DNA helix but compress the major groove. The compression of the DNA minor groove and not necessarily the curved trajectory of the DNA is, therefore, responsible for termination of DNA synthesis at the CTS by HIV-1 RT. This conclusion is consistent with interpretation of other biochemical data on the processivity of HIV-1 RT, based on the structure of a DNA-enzyme complex.

2-Aminopurine↗
PubMed1999

The kinetics of sigma subunit directed promoter recognition by E. coli RNA polymerase.

Time-resolved laser UV irradiation and controlled proteolysis have been used to study the sequential recognition of the lac UV5 promoter by Escherichia coli RNA polymerase. Local rearrangements in the DNA, the appearance of intimate protein-DNA contacts, and structural changes within the sigma subunit together provide specific signatures that define major species populated during this process. At 22 degreesC, a first closed complex is characterised by a transient conformational change in the sigma subunit and by a distortion in the -35 region. Subsequently, direct contacts at -34 and at positions -8, -5 and -3 on the non-template strand appear prior to DNA strand separation. The contact in the -35 consensus region involves only the sigma subunit. This intermediate possesses different structural parameters from that formed by quenching open complexes from 37 degreesC to 14 degreesC. Sigma thus appears as the principal partner acting during promoter recognition, a strongly coupled process involving two major intermediates only.

DNA, Bacterial↗
PubMed1998

Displacement of viral DNA termini from stable HIV-1 integrase nucleoprotein complexes induced by secondary DNA-binding interactions.

The human immunodeficiency virus type-1 (HIV-1) integrase is known to form a highly stable interaction with the termini of the linear, pre-integrated retroviral genome, where it catalyzes the 3'-OH processing and strand transfer processes required for their coordinated integration into host DNA. Here, we determine that the association of HIV-1 integrase with the viral DNA termini leads to the formation of two classes of nucleoprotein complexes with distinct properties in vitro. Both bound states are intrinsically stable and highly resistant to exonuclease digestion, but nonetheless they exhibit different stabilities in the presence of single-stranded polynucleotides. While a population of preassembled complexes tolerates elevated polynucleotide concentrations, the remainder forms an unstable ternary (integrase-substrate-polynucleotide) intermediate, leading to the rapid expulsion of the otherwise tightly bound substrate. The distribution of complexes between the two states is influenced by the preincubation time and temperature, increases in either of which favor the formation of the challenge-resistant species. Challenge-resistant complexes are formed more efficiently with Mn2+ than with Mg2+ and are sensitive to the length rather than the sequence of the DNA substrate. Due to the delayed appearance of the challenge-resistant form after the initial stable binding of the DNA substrate, our results may be indicative of a structural change in the preassembled complex which thereby modulates its response to exogenous DNA targets.

Base Sequence↗
PubMed1998

CRP interacts with promoter-bound sigma54 RNA polymerase and blocks transcriptional activation of the dctA promoter.

The cAMP receptor protein (CRP) is an activator of sigma70-dependent transcription. Analysis of the sigma54-dependent dctA promoter reveals a novel negative regulatory function for CRP. CRP can bind to two distant sites of the dctA promoter, sites which overlap the upstream activator sequences for the DctD activator. CRP interacts with Esigma54 bound at the dctA promoter via DNA loop formation. When the CRP-binding sites are deleted, CRP still interacts in a cAMP-dependent manner with the stable Esigma54 closed complex via protein-protein contacts. CRP is able to repress activation of the dctA promoter, even in the absence of specific CRP-binding sites. CRP affects both the final level and the kinetics of activation. The establishment of the repression and its release by the NtrC activator proceed via slow processes. The kinetics suggest that CRP favours a new form of closed complex which interconverts slowly with the classical closed intermediate. Only the latter is capable of interacting with an activator to form an open promoter complex. Thus, Esigma54 promoters are responsive to CRP, a protein unrelated to sigma54 activators, and the repression exerted is the direct result of an interaction between Esigma54 and the CRP-cAMP complex.

Bacterial Proteins↗
PubMed1998

The bacteriophage T4 AsiA protein: a molecular switch for sigma 70-dependent promoters.

The AsiA protein, encoded by bacteriophage T4, inhibits Esigma70-dependent transcription at bacterial and early-phage promoters. We demonstrate that the inhibitory action of AsiA involves interference with the recognition of the -35 consensus promoter sequence by host RNA polymerase. In vitro experiments were performed with a C-terminally labelled sigma factor that is competent for functional holoenzyme reconstitution. By protease and hydroxyl radical protein footprinting, we show that AsiA binds region 4.2 of sigma70, which recognizes the -35 sequence. Direct interference with the recognition of the promoter at this locus is supported by two parallel experiments. The stationary-phase sigma factor containing holoenzyme, which can initiate transcription at promoters devoid of a -35 region, is insensitive to AsiA inhibition. The recognition of a galP1 promoter by Esigma70 is not affected by the presence of AsiA. Therefore, we conclude that AsiA inhibits transcription from Escherichia coli and T4 early promoters by counteracting the recognition of region 4.2 of sigma70 with the -35 hexamer.

Amino Acid Sequence↗
PubMed1997

Structural kinetics of transcription activation at the malT promoter of Escherichia coli by UV laser footprinting.

We have studied the kinetics of transcriptional initiation and activation at the malT and malTp1 promoters of Escherichia coli using UV laser footprinting. Contrary to previous studies and because of the very rapid signal acquisition by this technique, we can obtain structural information about true reaction intermediates of transcription initiation. The consequences of adding a transcriptional activator, the cAMP receptor protein/cAMP complex (CRP), are monitored in real time, permitting us to assign specific interactions to the activation of discrete steps in transcription initiation. Direct protein-protein contacts between CRP and the RNA polymerase appeared very rapidly, followed by DNA melting around the -10 hexamer. CRP slightly increased the rate of this isomerization reaction but, more importantly, favored the establishment of additional contacts between the DNA upstream of the CRP binding site and RNA polymerase subsequent to open complex formation. These contacts make a major contribution to transcriptional activation by stabilizing open forms of the promoter complex, thereby indirectly accelerating promoter escape. The ensemble of the kinetic, structural signals demonstrated directly that CRP exerts most of its activating effects on the late stages of transcriptional initiation at the malT promoter.

Bacterial Proteins↗