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Biomedical subjects

A Stasiak

Publications and source records attributed to A Stasiak.

At least 91 records · Page 5Linked to original sources
PubMed1985

Duplex DNA knots produced by Escherichia coli topoisomerase I. Structure and requirements for formation.

We investigated systematically the knotting of nicked circular duplex DNA by Escherichia coli topoisomerase I. Agarose gel electrophoresis of knots forms a ladder of DNA bands. Each rung is made up of a variety of knots with the same number of nodes, or segment crossings; knots in adjacent rungs differ by one node. We extended the technique of electron microscopy of recA protein-coated DNA to the visualization of the complex knots tied by topoisomerase I. The striking result is that the enzyme produces every knot theoretically possible. The requirement for excess enzyme to form complex knots suggests a role for topoisomerase I in contorting the DNA in addition to promoting strand passage. We conclude that nodes formed are equally likely to be positive or negative and that topoisomerase I can pass DNA strands through a transient enzyme-generated break without regard to orientation of the passing strand. The results are interpreted in terms of a formulation for the topological requirements for knotting.

Bacteriophage phi X 174↗
PubMed1985

The stereostructure of knots and catenanes produced by phage lambda integrative recombination: implications for mechanism and DNA structure.

We studied the mechanism of recombination by determining the structure of the products of the phage lambda Int system. Electron microscopy of RecA-coated products revealed only knots and catenanes containing a regular right-handed spiral structure. The structure and distribution of products establish that the recombination sites pair by essentially random collision, rather than by tracking. However, the distribution also indicates that the binding of the enzyme must introduce nonrandom components into the reaction and stabilize at least two additional supercoils that become links in the product. Moreover, the regularity of the structures indicates that the strand exchange is accomplished in a very simple way, introducing only a single link into the product. All other links result from the direct conversion of substrate supercoils into knot and catenane links. These supercoils must be in a right-handed, braided form, rather than solenoidally wound as in nucleosomes.

Bacteriophage lambda↗
PubMed1983

Torsional stress induces left-handed helical stretches in DNA of natural base sequence: circular dichroism and antibody binding.

Above a threshold of torsional stress, the c.d. spectrum of covalently closed circular DNA of natural base sequence acquires a Z-like contribution and antibodies raised against Z-DNA are bound. Mapping of the antibody binding sites by electron microscopy reveals sites which correlate with stretches enriched in alternating purine-pyrimidine sequences and GC base pairs.

Animals↗
PubMed1982

The helicity of DNA in complexes with recA protein.

The RecA protein of Escherichia coli is involved in recombination (for review see ref. 1). The protein binds transiently to double-stranded DNA in the presence of ATP. In the presence of ATP gamma S, a non-hydrolysable analogue of ATP, recA-DNA complexes are stable. Duplex DNA in these complexes is stretched by a factor 1.5 (ref. 4), and the complexes appear in the electron microscope as helical filaments with a pitch of approximately 100 A and 6.2 recA units per turn covering 18.6 base pairs (bp). RecA crystals have a space group of similar helical parameters. In order to understand the function of recA, it is necessary to describe the conformation of the DNA in the recA complex. Using a topological method, the present work determines the helicity of DNA in the complex. We find that the DNA helix follows the protein helix visible in the electron microscope and has 18.6 bp per turn, which corresponds to an unwinding of the DNA double helix by 15 degrees per bp.

Bacterial Proteins↗
PubMedDate not supplied

RecA-DNA helical filaments in genetic recombination.

Paul Howard-Flanders et al proposed a molecular model of RecA-mediated recombination reaction six years ago. How does this model stand at present? In answering this question, we focus on two leading ideas of the original model, namely the proposal of the coaxial arrangement of the aligned DNA molecules within helical RecA filaments and the proposal of the ATP independence of the pairing stage of the recombination reaction. Results obtained after the model was proposed are reviewed and compared with these original assumptions and postulates of the model. EM visualization of recombining DNA molecules, studies of the energetics of the RecA-mediated recombination reaction and biochemical analysis of deproteinized joint molecules are fully consistent with a triple-stranded DNA arrangement during the RecA-mediated recombination reaction and demonstrate the ATP independence of the pairing stage of the reaction.

Adenosine Triphosphate↗
PubMedDate not supplied

Determination of the absolute handedness of knots and catenanes of DNA.

DNA winds about itself in a right-handed or left-handed fashion at several structural levels. The double helix is generally right-handed and is given a (+) sign by convention, whereas supercoiling of the helix axis is always (-) in the cell. The winding in higher -order forms such as knots and catenanes is unknown, and this has impeded elucidation of the mechanisms of their formation and resolution by replication, recombination and topoisomerase action. We introduce here a procedure for determining the handedness of DNA winding by inspection of electron micrographs of DNA molecules coated with Escherichia coli RecA protein. We demonstrate the validity of the method and show that DNA topoisomerase I of E. coli generates an equal mixture of (+) and (-) duplex DNA knots, and that one product of recombination by resolvase of transposon Tn3 (refs 8, 9) is a catenane of uniquely (+) sign.

Bacterial Proteins↗