Oligomerization and ATP stimulate condensin-mediated DNA compaction.

Large-scale chromatin remodeling during mitosis is catalyzed by a heteropentameric enzyme known as condensin. The DNA-organizing mechanism of condensin depends on the energy of ATP hydrolysis but how this activity specifically promotes proper compaction and segregation of chromosomes during mitosis remains poorly understood. Purification of budding yeast condensin reveals that it occurs not only in the classical heteropentameric "monomer" form, but that it also adopts much larger configurations consistent with oligomerization. We use a single-DNA magnetic tweezers assay to study compaction of DNA by yeast condensin, with the result that only the multimer shows ATP-enhanced DNA-compaction. The compaction reaction involves step-like events of 200 nm (600 bp) size and is strongly suppressed by forces above 1 pN, consistent with a loop-capture mechanism for initial binding and compaction. The compaction reactions are largely insensitive to DNA torsional stress. Our results suggest a physiological role for oligomerized condensin in driving gradual chromatin compaction by step-like and slow "creeping" dynamics consistent with a loop-extrusion mechanism.

Crossover-site sequence and DNA torsional stress control strand interchanges by the Bxb1 site-specific serine recombinase.

DNA segment exchange by site-specific serine recombinases (SRs) is thought to proceed by rigid-body rotation of the two halves of the synaptic complex, following the cleavages that create the two pairs of exchangeable ends. It remains unresolved how the amount of rotation occurring between cleavage and religation is controlled. We report single-DNA experiments for Bxb1 integrase, a model SR, where dynamics of individual synapses were observed, using relaxation of supercoiling to report on cleavage and rotation events. Relaxation events often consist of multiple rotations, with the number of rotations per relaxation event and rotation velocity sensitive to DNA sequence at the center of the recombination crossover site, torsional stress and salt concentration. Bulk and single-DNA experiments indicate that the thermodynamic stability of the annealed, but cleaved, crossover sites controls ligation efficiency of recombinant and parental synaptic complexes, regulating the number of rotations during a breakage-religation cycle. The outcome is consistent with a 'controlled rotation' model analogous to that observed for type IB topoisomerases, with religation probability varying in accord with DNA base-pairing free energies at the crossover site. Significantly, we find no evidence for a special regulatory mechanism favoring ligation and product release after a single 180° rotation.

Arginine as a general acid catalyst in serine recombinase-mediated DNA cleavage.

Members of the serine family of site-specific DNA recombinases use an unusual constellation of amino acids to catalyze the formation and resolution of a covalent protein-DNA intermediate. A recent high resolution structure of the catalytic domain of Sin, a particularly well characterized family member, provided a detailed view of the catalytic site. To determine how the enzyme might protonate and stabilize the 3'O leaving group in the strand cleavage reaction, we examined how replacing this oxygen with a sulfur affected the cleavage rate by WT and mutant enzymes. To facilitate direct comparison of the cleavage rates, key experiments used suicide substrates that prevented religation after cleavage. The catalytic defect associated with mutation of one of six highly conserved arginine residues, Arg-69 in Sin, was partially rescued by a 3' phosphorothiolate substrate. We conclude that Arg-69 has an important role in stabilizing the 3'O leaving group and is the prime candidate for the general acid that protonates the 3'O, in good agreement with the position it occupies in the high resolution structure of the active site of Sin.

Structural basis for catalytic activation of a serine recombinase.

Sin resolvase is a site-specific serine recombinase that is normally controlled by a complex regulatory mechanism. A single mutation, Q115R, allows the enzyme to bypass the entire regulatory apparatus, such that no accessory proteins or DNA sites are required. Here, we present a 1.86 Å crystal structure of the Sin Q115R catalytic domain, in a tetrameric arrangement stabilized by an interaction between Arg115 residues on neighboring subunits. The subunits have undergone significant conformational changes from the inactive dimeric state previously reported. The structure provides a new high-resolution view of a serine recombinase active site that is apparently fully assembled, suggesting roles for the conserved active site residues. The structure also suggests how the dimer-tetramer transition is coupled to assembly of the active site. The tetramer is captured in a different rotational substate than that seen in previous hyperactive serine recombinase structures, and unbroken crossover site DNA can be readily modeled into its active sites.