Quantitative analysis of chromosome condensation in fission yeast.
Petrova, B., Dehler, S., Kruitwagen, T., Heriche, J.K., Miura, K. & Haering, C.H.
Mol Cell Biol. 2013 Mar;33(5):984-98. doi: 10.1128/MCB.01400-12. Epub 2012 Dec21.
Chromosomes undergo extensive conformational rearrangements in preparation for their segregation during cell divisions. Insights into the molecular mechanisms behind this still poorly understood condensation process require the development of new approaches to quantitatively assess chromosome formation in vivo. In this study, we present a live-cell microscopy-based chromosome condensation assay in the fission yeast Schizosaccharomyces pombe. By automatically tracking the three-dimensional distance changes between fluorescently marked chromosome loci at high temporal and spatial resolution, we analyze chromosome condensation during mitosis and meiosis and deduct defined parameters to describe condensation dynamics. We demonstrate that this method can determine the contributions of condensin, topoisomerase II, and Aurora kinase to mitotic chromosome condensation. We furthermore show that the assay can identify proteins required for mitotic chromosome formation de novo by isolating mutants in condensin, DNA polymerase epsilon, and F-box DNA helicase I that are specifically defective in pro-/metaphase condensation. Thus, the chromosome condensation assay provides a direct and sensitive system for the discovery and characterization of components of the chromosome condensation machinery in a genetically tractable eukaryote.
A FlAsH-Based Cross-Linker to Study Protein Interactions in Living Cells.
Rutkowska, A., Häring, C.H. & Schultz, C.
Angew Chem Int Ed Engl. 2011 Dec 23;50(52):12655-8. doi:10.1002/anie.201106404. Epub 2011 Nov 16.
As you like it: xCrAsH, a dimeric derivative of the arsenical compound FlAsH, enables the highly specific, covalent cross-linking of two proteins containing a 12 amino acid peptide tag. This inducible and (by addition of dithiols) reversible system can be used to detect and manipulate protein-protein interactions both in vitro and in living cells.
Condensin structures chromosomal DNA through topological links.
Cuylen, S., Metz, J. & Häring, C.H.
Nat Struct Mol Biol. 2011 Jul 17;18(8):894-901. doi: 10.1038/nsmb.2087.
The multisubunit condensin complex is essential for the structural organization of eukaryotic chromosomes during their segregation by the mitotic spindle, but the mechanistic basis for its function is not understood. To address how condensin binds to and structures chromosomes, we have isolated from Saccharomyces cerevisiae cells circular minichromosomes linked to condensin. We find that either linearization of minichromosome DNA or proteolytic opening of the ring-like structure formed through the connection of the two ATPase heads of condensin's structural maintenance of chromosomes (SMC) heterodimer by its kleisin subunit eliminates their association. This suggests that condensin rings encircle chromosomal DNA. We further show that release of condensin from chromosomes by ring opening in dividing cells compromises the partitioning of chromosome regions distal to centromeres. Condensin hence forms topological links within chromatid arms that provide the arms with the structural rigidity necessary for their segregation.
The cohesin ring concatenates sister DNA molecules.
Häring, C.H., Farcas, A.M., Arumugam, P., Metson, J. & Nasmyth, K.
Nature. 2008 Jul 17;454(7202):297-301. Epub 2008 Jul 2.
Sister chromatid cohesion, which is essential for mitosis, is mediated by a multi-subunit protein complex called cohesin. Cohesin's Scc1, Smc1 and Smc3 subunits form a tripartite ring structure, and it has been proposed that cohesin holds sister DNA molecules together by trapping them inside its ring. To test this, we used site-specific crosslinking to create chemical connections at the three interfaces between the three constituent polypeptides of the ring, thereby creating covalently closed cohesin rings. As predicted by the ring entrapment model, this procedure produced dimeric DNA-cohesin structures that are resistant to protein denaturation. We conclude that cohesin rings concatenate individual sister minichromosome DNA molecules.