Biochemical analysis of the yeast condensin Smc2/4 complex: an ATPase that promotes knotting of circular DNA.
To better understand the contributions that the structural maintenance of chromosome proteins (SMCs) make to condensin activity, we have tested a number of biochemical, biophysical, and DNA-associated attributes of the Smc2p-Smc4p pair from budding yeast. Smc2p and Smc4p form a stable heterodimer, the "Smc2/4 complex," which upon analysis by sedimentation ... equilibrium appears to reversibly self-associate to form heterotetramers. Individually, neither Smc2p nor Smc4p hydrolyzes ATP; however, ATPase activity is recovered by equal molar mixing of both purified proteins. Hydrolysis activity is unaffected by the presence of DNA. Smc2/4 binds both linearized and circular plasmids, and the binding appears to be independent of adenylate nucleotide. High mole ratios of Smc2/4 to plasmid promote a geometric change in circular DNA that can be trapped as knots by type II topoisomerases but not as supercoils by a type I topoisomerase. Binding titration analyses reveal that two Smc2/4-DNA-bound states exist, one disrupted by and one resistant to salt challenge. Competition-displacement experiments show that Smc2/4-DNA-bound species formed at even high protein to DNA mole ratios remain reversible. Surprisingly, only linear and supercoiled DNA, not nicked-circular DNA, can completely displace Smc2/4 prebound to a labeled, nicked-circular DNA. To explain this geometry-dependent competition, we present two models of DNA binding by SMCs in which two DNA duplexes are captured within the inter-coil space of an Smc2/4 heterodimer. Based on these models, we propose a DNA displacement mechanism to explain how differences in geometry could affect the competitive potential of DNA.
Mesh Terms:
Adenosine Triphosphatases, Adenosine Triphosphate, Animals, Binding, Competitive, Carrier Proteins, Chromosomal Proteins, Non-Histone, DNA, DNA, Circular, Dimerization, Dose-Response Relationship, Drug, Electrophoresis, Polyacrylamide Gel, Genetic Complementation Test, Hydrolysis, Kinetics, Models, Molecular, Nuclear Proteins, Plasmids, Protein Binding, Protein Conformation, Saccharomyces cerevisiae Proteins, Ultraviolet Rays, Xenopus
Adenosine Triphosphatases, Adenosine Triphosphate, Animals, Binding, Competitive, Carrier Proteins, Chromosomal Proteins, Non-Histone, DNA, DNA, Circular, Dimerization, Dose-Response Relationship, Drug, Electrophoresis, Polyacrylamide Gel, Genetic Complementation Test, Hydrolysis, Kinetics, Models, Molecular, Nuclear Proteins, Plasmids, Protein Binding, Protein Conformation, Saccharomyces cerevisiae Proteins, Ultraviolet Rays, Xenopus
J. Biol. Chem.
Date: Jul. 11, 2003
PubMed ID: 12719426
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