Biomedical subjects

R J Reece

Publications and source records attributed to R J Reece.

5 recordsLinked to original sources

PubMed1991

The C-terminal domain of the Escherichia coli DNA gyrase A subunit is a DNA-binding protein.

We have constructed a clone which over-produces a 33 kDa protein representing the C-terminal portion of the Escherichia coli DNA gyrase A subunit. This protein has no enzymic activity of its own, but will form a complex with a 64 kDa protein (representing the N-terminal part of the A subunit) and the gyrase B subunit, that will efficiently catalyse DNA supercoiling. We show that the 33 kDa protein can bind to DNA on its own in a manner which induces positive supercoiling of the DNA. We propose that the 33 kDa protein represents a domain of the gyrase A subunit which is involved in the wrapping of DNA around DNA gyrase.

Base Sequence↗

PubMed1991

Probing the limits of the DNA breakage-reunion domain of the Escherichia coli DNA gyrase A protein.

In a previous report (Reece, R. J., and Maxwell, A. (1989) J. Biol. Chem. 264, 19648-19653) we showed that treatment of the Escherichia coli DNA gyrase A protein with trypsin generates two stable fragments. The N-terminal 64-kDa fragment supports DNA supercoiling, while the C-terminal 33-kDa fragment shows no enzymic activity. We proposed that the 64-kDa fragment represents the DNA breakage-reunion domain of the A protein. We have now engineered the gyrA gene such that the 64-kDa protein is generated as a gene product. The properties of this protein confirm the findings of the experiments with the 64-kDa tryptic fragment. We have also generated a series of deletions of the gyrA gene such that C-terminal and N-terminal truncated versions of the A protein are produced. The smallest of the N-terminal fragments found to be able to carry out the DNA breakage-reunion reaction is GyrA(1-523). The cleavage reaction mediated by this protein occurs with equal efficacy as that performed by the intact GyrA protein. Deletion of the N-terminal 6 amino acids from either the A protein or these deletion derivatives has no effect on enzymic activity, while deletion of the N-terminal 69 amino acids completely abolishes the DNA breakage-reunion reaction. Therefore the smallest GyrA protein we have found that will perform DNA breakage and reunion is GyrA(7-523). A model is proposed for the domain organization of the gyrase A protein.

Cloning, Molecular↗

PubMed1991

DNA gyrase: structure and function.

DNA gyrase is an essential bacterial enzyme that catalyzes the ATP-dependent negative super-coiling of double-stranded closed-circular DNA. Gyrase belongs to a class of enzymes known as topoisomerases that are involved in the control of topological transitions of DNA. The mechanism by which gyrase is able to influence the topological state of DNA molecules is of inherent interest from an enzymological standpoint. In addition, much attention has been focused on DNA gyrase as the intracellular target of a number of antibacterial agents as a paradigm for other DNA topoisomerases. In this review we summarize the current knowledge concerning DNA gyrase by addressing a wide range of aspects of the study of this enzyme.

Anti-Bacterial Agents↗

PubMed1990

Preliminary crystallographic analysis of the breakage-reunion domain of the Escherichia coli DNA gyrase A protein.

The 64 x 10(3) Mr N-terminal breakage-reunion domain of the Escherichia coli DNA gyrase A protein was purified from an over-expressing strain. When complexed with the gyrase B protein, this truncated A protein has all of the enzymic properties of the full-length counterpart, although with reduced efficiency in some cases. The 64 x 10(3) Mr protein has been crystallized in several forms, a number of which were too small for crystallographic analysis. However, two forms grew to sufficient size for preliminary X-ray analysis. Both forms were tetragonal with a primitive lattice. One form (type I) had cell dimensions of a = b = 170 A, c = 145 A a space group of either P41212 (P43212) or P42212, and diffracted to 6 A resolution. The type II crystals had cell dimensions of a = b = 177 A, c = 175 A, a space group of P41212 (P43212) or P42212, and diffracted to at least 4.5 A resolution. Both crystal forms apparently contained four subunits (possibly a tetramer) in the asymmetric unit. We are attempting to increase the size and quality of these crystals.

DNA Topoisomerases, Type II↗

PubMed1989

Tryptic fragments of the Escherichia coli DNA gyrase A protein.

Treatment of the Escherichia coli DNA gyrase A protein with trypsin generates two large fragments which are stable to further digestion. The molecular masses of these fragments are 64 and 33 kDa, and they are shown to be derived from the N terminus and the C terminus of the A protein, respectively. These fragments could represent structural and/or functional domains within the A subunit of DNA gyrase. The trypsin-cleaved A protein (A'), in combination with the B subunit of gyrase, can support ATP-dependent supercoiling of relaxed DNA and other reactions of DNA gyrase. The isolated 64-kDa fragment will also catalyse DNA supercoiling in the presence of the B protein, but the 33-kDa fragment shows no enzymic activities. We conclude that the N-terminal 64-kDa fragment represents the DNA breakage/reunion domain of the A protein, while the 33-kDa fragment may contribute to the stability of the gyrase-DNA complex.

DNA Topoisomerases, Type II↗