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G Chaconas

Publications and source records attributed to G Chaconas.

At least 37 records · Page 2Linked to original sources
PubMed1993

Site-specific HU binding in the Mu transpososome: conversion of a sequence-independent DNA-binding protein into a chemical nuclease.

HU is a small, basic, sequence-independent DNA-binding protein capable of engineering DNA deformations required for the formation of higher order nucleoprotein structures. One such complex is the Mu Type 1 transpososome, where the ends of Mu are stably synapsed by a tetramer of Mu A and cleaved at their 3' ends. HU is believed to play a critical role in transpososome assembly, which requires the communication of the two Mu ends and the transpositional enhancer. Although footprinting studies have clearly defined the DNA regions bound by Mu A, no protection could be ascribed to the HU protein by DNAse I, MPE.Fe(II) or hydroxyl radical methods (Lavoie et al. 1991). To directly probe HU interactions with the transpososome DNA, we have coupled HU to a DNA cleavage reagent, iron-EDTA, and report here the first HU "footprint". HU-nuclease cleavage was detectable at specific sites within an 83-bp spacer DNA separating the left-end-most L1 site from its neighboring L2 site. This HU binding was specific since it could not be competed with 10-fold excess supercoiled DNA. We postulate that HU promotes the formation of a tight DNA bend or loop in this region which facilitates the communication of Mu A monomers during complex assembly. This method may prove generally useful for the localization of sequence-independent DNA-binding proteins on DNA and within higher oder nucleoprotein structures.

Bacterial Proteins↗
PubMed1992

Role of the A protein-binding sites in the in vitro transposition of mu DNA. A complex circuit of interactions involving the mu ends and the transpositional enhancer.

To investigate the role of the A protein-binding sites at the Mu ends in the DNA strand transfer reaction, we constructed mutant mini-Mu molecules in which these sites were deleted (L3 or R3) or substituted (L2 or R2) to conserve the spacing arrangements at the adjacent sites. The single site mutants are poor substrates for phosphodiester bond hydrolysis at the Mu ends in Type 1 reactions in the absence of Escherichia coli integration host factor (IHF). Addition of IHF to the reaction stimulates Type 1 cleavage more than 10 times for the delta-R3, delta-L3, S-L2 mutants and more than five times in the case of the S-R2 mutant under alternate conditions. The site of IHF stimulation resides within the transpositional enhancer which implicates the end-binding sites L2, L3, R2, and R3 in interactions with the enhancer. At least two of the L2, L3, and R3 sites are required for proficient reaction in the presence of IHF. By combining the single site mutants with O1 or O2 partially deleted enhancer elements, we have tentatively localized some of the interactions to each side of the functional enhancer revealing a complex circuit of end-enhancer interactions. The R3 site is suggested to be involved in interactions only with O2 and the L3 site only with O1. The data also suggest the possibility that L2 and R2 may be involved in interactions with both O1 and O2. Finally, our working model predicts that the L3-O1 and R3-O2 interactions may be required contacts for discriminating between the Mu left and right ends in transpososome formation.

Bacterial Proteins↗
PubMed1992

Flanking host sequences can exert an inhibitory effect on the cleavage step of the in vitro mu DNA strand transfer reaction.

The effect of flanking host sequences on the cleavage step of the in vitro Mu DNA strand transfer reaction was investigated. Insertion of a mini-Mu molecule into certain sites in pUC19 results in insertions that demonstrate a decreased ability to form Type 1 complexes in subsequent rounds of transposition. Similarly, changes in the flanking host sequences directly adjacent to the Mu ends by in vitro mutagenesis can also result in Type 1-deficient mini-Mu molecules. Further examination of the inhibition revealed that Type 1 deficient mini-Mu molecules are capable of forming uncut synaptic complexes at normal levels but are compromised in their ability to serve as substrates for phosphodiester bond hydrolysis at the Mu ends. This cleavage defect can be overcome by addition of the Mu B protein and ATP to the reaction. Our data suggest that one of the roles of the B protein may be to provide a mechanism whereby Mu prophages with inhibitory flanking sequences can overcome this obstacle and avoid being trapped at unproductive locations.

Bacteriophage mu↗
PubMed1992

The Mu transpositional enhancer can function in trans: requirement of the enhancer for synapsis but not strand cleavage.

The phage Mu transpositional enhancer has been previously shown to stimulate the initial rate of the Mu DNA strand transfer reaction by a factor of 100. We now show that the Mu enhancer can function in trans on an unlinked DNA molecule. This activity is greatly facilitated by the presence of a free DNA end proximal to the enhancer element. Function of the enhancer in trans does not alter either the requirement for donor DNA supercoiling or for the two Mu ends to be in their proper orientation on the donor plasmid. An important consequence of these findings is that we have been able to evaluate directly the step in the transposition reaction for which the enhancer is required. We show that the role of the enhancer is limited to promoting productive synapsis; efficient strand cleavage can occur in the absence of the enhancer.

Bacteriophage mu↗
PubMed1992

Mechanistic aspects of DNA transposition.

The past year has seen a number of important advances in our understanding of the mechanisms of DNA transposition. The molecular details of the protein-protein, protein-DNA and chemical-reaction steps in several transposition systems have been revealed and have highlighted remarkable uniformity in some areas, ranging from bacterial to retroviral mechanisms.

Bacteriophage mu↗
PubMed1991

Stimulation of the Mu DNA strand cleavage and intramolecular strand transfer reactions by the Mu B protein is independent of stable binding of the Mu B protein to DNA.

Interactions between the Mu A and Mu B proteins are important in the early steps of the in vitro transposition of a mini-Mu plasmid. We have examined these interactions by assaying Mu B stimulation of Mu A-mediated strand cleavage and strand transfer reactions. We have previously shown that in the presence of ATP the Mu B protein can stimulate the Mu A-directed cleavage reaction of mini-Mu plasmids carrying a terminal base pair mutation (Surette, M.G., Harkness, T., and Chaconas, G. (1991) J. Biol. Chem. 266, 3118-3124). Here we demonstrate that in the absence of a non-Mu DNA target molecule the Mu B protein stimulates intramolecular integration of a mini-Mu in an ATP-dependent fashion. Furthermore, modification of the Mu B protein with N-ethylmaleimide severely compromises the ability of B to form a stable complex with DNA; however, the modified protein stimulates the strand cleavage and intramolecular strand transfer reactions as efficiently as the untreated protein. These results indicate that the Mu B protein is capable of stimulating the Mu A protein through direct interaction in the absence of stable Mu B-DNA complex formation. Our results increase the spectrum of Mu B protein activities and uncouple the stimulatory properties of the Mu B protein from stable DNA binding but not the ATP cofactor requirement.

Adenosine Triphosphatases↗
PubMed1991

Stimulation of the Mu A protein-mediated strand cleavage reaction by the Mu B protein, and the requirement of DNA nicking for stable type 1 transpososome formation. In vitro transposition characteristics of mini-Mu plasmids carrying terminal base pair mutations.

We have examined the effects of a T----C point mutation at the terminal nucleotide of the Mu ends in a mini-Mu plasmid on the early steps in the in vitro transposition reaction. These mutations inhibit the introduction of nicks at the Mu ends in a reaction with Mu A, HU, and integration host factor proteins. The presence of the point mutation at either the left end or the right end is sufficient to block the nicking reaction at both ends, indicating that the reaction is normally concerted. Addition of Mu B and ATP, however, dramatically stimulates the reaction of mutant mini-Mu plasmids carrying the mutation at one end but not at both ends. The data suggest that the Mu B protein mediates its effect through direct interaction with Mu A and that Mu B may play a role in an earlier step in the transposition process than previously proposed. In the presence of Mu B, two products are observed with the left end or right end mutant mini-Mu plasmids, a normal protein-DNA intermediate (Type 1 complex) which contains nicks at both Mu ends and an abortive product composed of free relaxed plasmid which is nicked only at the wild-type end. Furthermore, stable protein-DNA complexes characteristic of the first step in the in vitro transposition reaction are not observed in the absence of nicking or when only one end is a nicked; the introduction of nicks at both Mu ends is a prerequisite for stable transpososome assembly.

Adenosine Triphosphate↗
PubMed1991

Structural aspects of a higher order nucleoprotein complex: induction of an altered DNA structure at the Mu-host junction of the Mu type 1 transpososome.

The Mu in vitro strand transfer reaction proceeds via two stable higher order nucleoprotein complexes, the Type 1 and Type 2 transpososomes. The Mu A protein is responsible for the structural and functional integrity of the Type 1 transpososome. We have investigated the quaternary structure of the Mu A protein within this complex by chemical cross-linking experiments and found that the basic structural unit is an A tetramer. Three Mu A binding sites in the transpososome are protected by DNase I footprinting: the outermost A binding sites L1 and R1, as well as R2. Genetic evidence is also presented which corroborates this result. Efficient formation of Type 1 complexes occurs in mini-Mus with the L3 or R3 sites deleted or when the L2 site has been substituted; but no reaction occurs in the absence of R2. The protection at the L1 and R1 sites extends 12-13 bp beyond the Mu-host junctions as seen by DNase I and methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] foot-printing, indicating Mu A contacts with the flanking host sequences in the transpososome but not on linear DNA; furthermore, hydroxyl radical footprinting shows an unprecedentedly large enhancement on the continuous strand, 2 bp beyond the nick site outside the Mu right end, which suggests that an altered DNA structure is induced upon Type 1 complex formation.

Bacterial Proteins↗
PubMed1990

Immunoelectron microscopic analysis of the A, B, and HU protein content of bacteriophage Mu transpososomes.

Stable protein-DNA complexes or transpososomes mediate the Mu DNA strand transfer reaction in vitro (Surette, M. G., Buch, S. J., and Chaconas, G. (1987) Cell 49, 253-262; Craigie, R., and Mizuuchi, K. (1987) Cell 51, 493-501). Formation of the Type 1 complex, an intermediate in the strand transfer reaction, requires the Mu A and Escherichia coli HU proteins. Generation of the Type 2 complex, in which the Mu ends have been covalently linked to the target DNA, requires the Mu B protein, ATP, and target DNA in addition to A and HU. The protein content of these higher order synaptic complexes has been studied by immunoelectron microscopy using protein A-colloidal gold conjugates to visualize antibody-bound complexes. Under our in vitro transposition conditions, Type 1 complexes were found to contain A and HU; in addition, Type 2 complexes contained Mu B. However, both the HU and the Mu B protein were found to be loosely associated and could be quantitatively removed from the nucleoprotein core of both complexes by incubation in 0.5 M NaCl. Depletion of HU from the Type 1 complex did not affect the ability of this complex to be converted into the strand-transferred product. Hence, the indispensable role of the HU protein in the Mu DNA strand transfer reaction is limited to the formation of the Type 1 transpososome.

Bacterial Proteins↗
PubMed1989

Secondary structural features of the bacteriophage Mu-encoded A and B transposition proteins.

The role of the bacteriophage Mu-encoded A and B proteins is to direct the transposition of Mu DNA. These are the first active DNA transposition proteins to have been purified and their mechanism of action at the biochemical level is under intensive study. Structural studies on these proteins, however, have lagged behind their biochemical characterization. We report here near- and far-u.v. c.d. spectra for these proteins and their secondary structural features derived from these data. The Mu A protein appears to be composed of primarily beta-sheet (40%) with 24% alpha-helix, 9% beta-turn and 27% random coil. In contrast, the Mu B protein contains 55% alpha-helix with only 13% beta-sheet and 3+ beta-turn and 29% random coil. The near-u.v. c.d. spectrum of the A protein was not unusual; however, the profile of the B protein suggested either buried or restricted chromophores within the protein or short-range interactions between aromatic residues.

Circular Dichroism↗
PubMed1989

A protein factor which reduces the negative supercoiling requirement in the Mu DNA strand transfer reaction is Escherichia coli integration host factor.

We have examined the supercoiling requirement for the in vitro Mu DNA strand transfer reaction and found that optimal efficiency requires a high level (sigma = -0.06) of donor plasmid superhelicity. At in vivo levels of supercoiling (sigma = -0.025) the reaction does not occur. Using an unreactive donor plasmid with a near physiological level of supercoiling, we identified an Escherichia coli protein factor which has the novel property of reducing the donor plasmid supercoiling requirement for the in vitro Mu DNA strand transfer reaction by 40%. This protein, which we named supercoiling relief factor was purified to near homogeneity and found to be identical to integration host factor (IHF), a protein known to induce site specific bends in DNA. The dramatic reduction in the supercoiling requirement was promoted by about 1.5 IHF dimers/donor substrate molecule. At these low levels of IHF, the HU requirement for the reaction was also reduced; a synergistic effect of the two proteins resulted in a greater than 10-fold stimulation of the reaction under appropriate conditions. Furthermore, at high concentrations of IHF, HU could be completely eliminated from the reaction.

Bacterial Proteins↗
PubMed1989

Action at a distance in Mu DNA transposition: an enhancer-like element is the site of action of supercoiling relief activity by integration host factor (IHF).

The first committed step in the in vitro strand transfer reaction of a mini-Mu donor molecule is the formation of a Type 1 complex in which the Mu ends are held together in a non-covalent protein-DNA complex. Efficient formation of this complex at high levels of donor supercoiling (sigma approximately -0.06) requires the Mu A and Escherichia coli HU proteins. At in vivo levels of supercoiling, efficient reaction also requires E. coli integration host factor (IHF). We demonstrate that this supercoiling relief activity of IHF is mediated through an IHF binding site in the Mu early promoter region. This site is part of a larger enhancer-like element which includes operator 1 (01) and part of operator 2 (02) with the IHF site in between. The enhancer-like element stimulates the initial rate of the in vitro reaction 100-fold and acts in a distance-independent fashion. Inversion of the orientation of the element results in a total loss of enhancer activity in the absence of IHF. However, a 10-fold stimulation in the initial rate of reaction is induced by the addition of IHF. Furthermore, correct helical phasing between 01 and 02 is required for maximal activity. The results indicate that a specific geometrical configuration of the enhancer-like element, which includes a sharp bend between 01 and 02, is required for optimal induction of synapsis.

Bacterial Proteins↗
PubMed1987

Transpososomes: stable protein-DNA complexes involved in the in vitro transposition of bacteriophage Mu DNA.

We report that two types of stable protein-DNA complexes, or transpososomes, are generated in vitro during the Mu DNA strand transfer reaction. The Type 1 complex is an intermediate in the reaction. Its formation requires a supercoiled mini-Mu donor plasmid, Mu A and HU protein, and Mg2+. In the Type 1 complex the two ends of Mu are held together, creating a figure eight-shaped molecule with two independent topological domains; the Mu sequences remain supercoiled while the vector DNA is relaxed because of nicking. In the presence of Mu B protein, ATP, target DNA, and Mg2+, the Type 1 complex is converted into the protein-associated product of the strand transfer reaction. In this Type 2 complex, the target DNA has been joined to the Mu DNA ends held in the synaptic complex at the center of the figure eight. Supercoils are not required for the latter reaction.

Bacteriophage mu↗
PubMed1986

The bacteriophage Mu N gene encodes the 64-kDa virion protein which is injected with, and circularizes, infecting Mu DNA.

Upon infection of Escherichia coli with bacteriophage Mu, a 64-kDa protein is injected into the host cell along with the phage DNA. This protein is involved in circularizing the infecting Mu DNA (Harshey, R. M., and Bukhari, A. I. (1983) J. Mol. Biol. 167, 427-441; Puspurs, A. H., Trun, N. J., and Reeve, J. N. (1983) EMBO J. 2, 345-352). Its possible role in the integration of infecting Mu DNA and in the infection process remains to be established. To identify the source of this protein we have prepared antiserum to the protein purified from viral particles. We have shown that the antiserum is specific for the Mu N gene product. The antiserum has been used to immunologically screen a Mu DNA library cloned into an expression vector. Four clones have been shown to produce a protein of 64 kDa that is specifically bound by the antiserum. The only Mu gene common to all four clones is the N gene, as demonstrated by physical and genetic mapping. We have also demonstrated by peptide mapping that the cloned N gene product is identical to the 64-kDa protein found complexed with the injected Mu DNA.

Cloning, Molecular↗
PubMed1986

Electron microscopic analysis of in vitro transposition intermediates of bacteriophage Mu DNA.

Bacteriophage Mu is a highly efficient transposon and the only moveable element for which an in vitro transposition system has been reported. Recently, this system has been used by Craigie and Mizuuchi [Cell 41 (1985) 867-876] to identify and biochemically characterize intermediates in the transposition process. We have utilized the in vitro transposition system to generate intermediates in the transposition process and have analyzed these intermediates by electron-microscopic methods. Partial denaturation mapping has shown the intermediates to be theta-shaped structures in which the phi X174 target DNA is joined to the mini-Mu plasmid at the ends of the Mu genome. Our results are in agreement with the previous biochemical studies and the type of intermediate we observe is exactly what is predicted by the Shapiro model of transposition [Proc. Natl. Acad. Sci. USA 76 (1979) 1933-1937].

Bacteriophage mu↗
PubMed1985

Amplification and purification of the bacteriophage Mu encoded B transposition protein.

The A and B proteins encoded by the temperate bacteriophage Mu are involved in the high efficiency DNA transposition reaction which is the distinguishing feature of this phage. The genes encoding these early proteins were cloned in an expression vector under the control of the bacteriophage lambda leftward promoter. Under optimal conditions gpB was overproduced to account for 15% of the total cellular protein. The protein was purified to near homogeneity as determined by silver staining. Sequence analysis of the N terminus confirmed the identity of the purified protein as gpB. Proteolytic processing of the B protein does not occur at the amino terminus; the terminal methionine residue is quantitatively deformylated. The protein, which was found to be basic and a general DNA binding protein, was insoluble at low ionic strength in the absence, but not in the presence, of DNA. The B protein also displayed a tendency to aggregate at high ionic strength where it was soluble in the absence of DNA. In addition, the protein was characterized as to its amino acid composition and extinction coefficient at 280 nm. The purified protein is active in a soluble in vitro transposition-replication system.

Amino Acids↗