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B D Lavoie

Publications and source records attributed to B D Lavoie.

10 recordsLinked to original sources
PubMed2000

Mitotic chromosome condensation requires Brn1p, the yeast homologue of Barren.

In vitro studies suggest that the Barren protein may function as an activator of DNA topoisomerase II and/or as a component of the Xenopus condensin complex. To better understand the role of Barren in vivo, we generated conditional alleles of the structural gene for Barren (BRN1) in Saccharomyces cerevisiae. We show that Barren is an essential protein required for chromosome condensation in vivo and that it is likely to function as an intrinsic component of the yeast condensation machinery. Consistent with this view, we show that Barren performs an essential function during a period of the cell cycle when chromosome condensation is established and maintained. In contrast, Barren does not serve as an essential activator of DNA topoisomerase II in vivo. Finally, brn1 mutants display additional phenotypes such as stretched chromosomes, aberrant anaphase spindles, and the accumulation of cells with >2C DNA content, suggesting that Barren function influences multiple aspects of chromosome transmission and dynamics.

Cell Cycle Proteins↗
PubMed1999

Supercoiling-dependent site-specific binding of HU to naked Mu DNA.

Using HU chemical nucleases to probe HU-DNA interactions, we report here for the first time site-specific binding of HU to naked DNA. An unique feature of this interaction is the absolute requirement for negative DNA supercoiling for detectable levels of site-specific DNA binding. The HU binding site is the Mu spacer between the L1 and L2 transposase binding sites. Our results suggest recognition of an altered DNA structure which is induced by DNA supercoiling. We propose that recruitment of HU to this naked DNA site induces the DNA bending required for productive synapsis and transpososome assembly. Implications of HU as a supercoiling sensor with a potential in vivo regulatory role are discussed. Finally, using HU nucleases we have also shown that non-specific DNA binding by HU is stimulated by increasing levels of supercoiling.

Bacterial Proteins↗
PubMed1996

Anatomy of a flexer-DNA complex inside a higher-order transposition intermediate.

SUMMARY: Escherichia coli HU, a nonsequence-specific histone- and HMG-like DNA-binding protein, was chemically converted into a series of HU-nucleases with an iron-EDTA-based cleavage moiety positioned at 16 rationally selected sites. Specific DNA cleavage patterns from each of these HU-nucleases allowed us to determine the precise localization, stoichiometry, and orientation of HU binding in the Mu transpososome, a multiprotein structure that mediates the chemical reactions in DNA transposition. Correlation of the DNA cleavage data with the position of the cleavage moiety in the HU three-dimensional structure indicates the presence of a dramatic DNA bend, for which the bend center, direction, and magnitude were assessed. The data, which directly localize selected HU amino acids with respect to DNA in the transpososome, were used as constraints for computer-based molecular modeling to derive the first snapshot of an HU-DNA interaction.

Amino Acid Sequence↗
PubMed1994

A second high affinity HU binding site in the phage Mu transpososome.

The bacteriophage Mu in vitro transposition reaction proceeds through several higher order nucleoprotein intermediates (transpososomes). One of the requirements for complex assembly is the Escherichia coli sequence-independent DNA-binding protein, HU. This protein has an affinity for Mu transpososomes, which is at least 100 times greater than for supercoiled DNA (Lavoie and Chaconas, 1990). We have recently identified one such high affinity binding site at the Mu left end by converting HU into a chemical nuclease (Lavoie and Chaconas, 1993). Using immunoelectron microscopy, we now report high affinity HU binding to a region(s) distinct from the previously characterized left end site. HU can be removed from this region by a 0.5 M NaCl wash and subsequently reassembled into the complex with high efficiency. Furthermore, chemical modification of the Mu A protein in the Type 1 complex does not block HU reassembly into the transpososome; the high affinity HU binding observed is therefore unlikely to result from A-HU interactions. These findings are corroborated by the ability of eukaryotic HMG-1 to functionally replace HU in transpososome formation and to efficiently assemble into HU-depleted complexes. We propose that HU recognition of an altered DNA structure, rather than protein-protein interactions, mediates high affinity HU binding to Mu transpososomes.

Bacterial Proteins↗
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↗
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

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↗