Douglas Koshland Staff Member Department of Embryology Carnegie Institution of Washington Investigator Howard Hughes Medical Institute Adjunct Professor Department of Biology CMDB Graduate Program Faculty B.A. Haverford College Ph.D. Massachusetts Institute of Technology

[Research Interests] [Representative Publications]

RESEARCH INTERESTS

Research summary:

Through the analysis of factors that mediate chromosome condensation and sister chromatid cohesion during mitosis and meiosis, we hope to elucidate the underlying molecular mechanism of chromosome structure and the principles that govern chromosome evolution.

Chromosome Structure and Segregation

Higher-order chromosome structure is critical to the segregation of replicated chromosomes (sister chromatids) during cell division. First, sister chromatids are paired along their entire length, from the time of replication until their segregation in mitosis. Pairing is needed to establish a stable bipolar attachment of sister chromatids to microtubules emanating from opposite poles of the mitotic spindle. This bipolar attachment ensures that sister chromatids segregate from each other during anaphase into the newly forming daughter cells. In addition, the dissolution of pairing at the onset of chromosome segregation is a key regulatory step for both normal cell division and cell survival in response to environmentally induced damage.

Sister chromatids also condense during mitosis, which helps to resolve them into distinct domains, reducing their entanglement while they move during mitosis. Condensation also ensures that the mitotic chromosomes are less than half as long as the spindle, thus preventing the lagging ends of segregating chromosomes from crossing the plane of cell division and being cleaved by cytokinesis. The segregated chromosomes must then decondense to allow DNA replication in the next cell cycle.

Sister chromatid cohesion. Chromosomes undergo DNA replication to form two sister chromatids. Cohesion (green) occurs concomitantly with replication near the centromere and along the length of the chromatid arms. Sister chromatids condense and the spindle forms. Cohesion between sister chromatids sterically constrains the orientation of the centromere/kinetochore (red) so that they favor attachment to microtubules (blue lines) from opposite poles (blue boxes). At the onset of anaphase, cohesion is dissolved and sister chromatid segregation ensues. Note that although cohesion is dissolved, at least some of the factors (not shown) that mediate cohesion may remain on the chromatids for a significant portion of anaphase.

From Koshland, D.E., and Guacci, V. 2000. Current Opinion in Cell Biology 12:297—301. © 2000, with permission from Elsevier Science.

To gain new insights into the role of chromosome dynamics in the cell cycle and higher-order chromosome folding, we use the budding yeast as a model system. We, and others, have identified structural components of cohesion (cohesins) and condensation (condensins). Cohesins bind to 1-kilobase regions spaced at ~3-kb intervals along chromosome arms. At the centromere, binding is much denser and extends over tens of kilobases. Whether condensins also bind to specific regions is unclear. Both complexes contain Smc proteins, which are large proteins that have a head domain with ATPase activity at one end and a hinge domain at the other. These two domains are connected by an extensive coiled-coil domain. Smc proteins dimerize through contacts between the hinge domains at one end and the head domains at the other. The head domains also recruit non-Smc subunits. Smc complexes have now been implicated in many other aspects of DNA metabolism, including control of global gene expression, DNA repair, and homologous recombination. Although the exact molecular activity of Smc complexes is unclear, they are thought to tether together two different DNA strands or two different parts of one DNA strand.

To begin to understand the mechanism of tethering, we have initiated a genetic approach to trap intermediates in Smc-complex function and assembly. From these analyses, we have determined that cohesins establish cohesion through a two-step process—first binding to specific chromosomal addresses on one sister chromatid and then, by activation through an auxiliary factor, establishing cohesion with the second sister chromatid. Coupling this two-step process with DNA replication may provide the temporal and spatial control to ensure that cohesins cross-link only sister chromatids and not random chromatin. We believe an analogous two-step mechanism is likely to underlie the tethering activity of all Smc complexes.

Our previous studies showed that cohesins also regulate condensin activity to ensure proper chromosome folding. Our recent studies in meiosis show that condensins regulate the removal of a subset of cohesins by activating a master cell cycle kinase. Furthermore, our recent studies on DNA repair show that the MRX/MRN complex, a Smc-like complex, is required to load cohesins adjacent to the site of a break in the DNA strand. This crosstalk between condensins, cohesins, and MRX reveals a general necessity for communication between Smc complexes to modulate chromosome architecture.

We have also begun to investigate new biological functions for condensins and cohesins beyond their established function in chromosome segregation. During cell division, chromosomes must not only segregate properly but also remain intact. Recently we discovered in budding yeast, a novel role for cohesin in the repair of double-strand breaks in chromosomal DNA. We show that cohesins are loaded extensively around a double-strand break, generating a large domain (100 kb) that covers as much as one-third of the chromosomes. This large cohesin domain is assembled by extensive phosphorylation of the histones over the 100 kb. Cohesins are loaded around the phosphorylated histones by a specialized repair complex, cohesin-loading factors and chromatin-remodeling factors. These cohesins use the sister chromatid as a template to stimulate repair.

Recently we have begun to examine chromosome integrity in the context of genome evolution. Chromosome structure rapidly evolves between species. A common route to new speciation in plants results from the formation of hybrids. To begin to understand chromosome evolution during speciation, we are analyzing changes in chromosome structure in hybrids of yeast species, in collaboration with Yixian Zheng (HHMI, Carnegie Institution of Washington) and Maitreya Dunham (Princeton University). We show that evolved hybrid yeast exhibit complex changes in genome structure—including chromosome loss, large amplifications, and translocations—similar to the differences seen between species and between normal and malignant cells.

We have begun to manipulate genome structure as well as components of repair metabolism to test the contribution to genome integrity of disperse repetitive DNA, whole-genome duplication, sequence divergence, and protein buffering.

REPRESENTATIVE PUBLICATIONS

Ünal, E., Pauli, J.H.,  Koshland, D. 2007. DNA double-strand breaks trigger genome-wide sister chromatid cohesion through Eco1/ Ctf7. Science 317:245-248.

Yu, H.G. and Koshland, D. 2007. The aurora kinase Ipl1 maintains centromeric localization of PP2A to protect cohesin during meiosis. Journal of Cell Biology 176:911-918.

Milutinovich, M., Ünal, E., Ward, C., Skibbens, R.V., Koshland, D. 2007. A multi-step pathway for the establishment of sister chromatid cohesion . PLoS Genet 19:3:e12.

Noble, D., Kenna M.A., Dix, M., Skibbens, R.V., Ünal, E., and Guacci V. 2006. Intersection between the regulators of sister chromatid cohesion establishment and maintenance in budding yeast indicates a multi-step mechanism. Cell Cycle Nov 1:5.

Milutinovich, M., Ünal, E., Ward, C., Skibbens, R.V. and Koshland D. 2006. A multi-step pathway for the establishment of sister chromatid cohesion.

Huang, C.E., Milutinovich, M. and Koshland D. 2005. Rings, bracelet or snaps: fashionable alternatives for Smc complexes. Phil. Trans.: Biological Sciences 360:537-542.

Yu, H.G., and Koshland D. 2005. Chromosome morphogenesis: condensin-dependent cohesin removal during meiosis. Cell 123:397-407.

Weber, S.A., Gerton, J., Polancic, J.E., DeRisi, J.L, Koshland, D., Megee, P.C. 2004. The kinetochore is an enhancer of pericentric cohesin binding. PloS Biol. 2: E260.

Glynn, E.F., Megee, P.C., Yu, H.G., Mistrot, C., Unal, E., Koshland, D.E., DeRisi, J.L. and Gerton, J.L. 2004. Genome-wide mapping of the cohesin complex in the yeast Saccharomyces cerevisiae. PLoS Biol. 2:E259.

Unal, E., Arbel-Eden, A., Sattler, U., Shroff, R., Lichten, M., Haber, J.E. and Koshland, D. 2004. DNA damage response pathway uses histone modification to assemble a double-strand break specific cohesin domain. Molecular Cell 16:991-1002.

Lavoie, B.D., Hogan, E. and Koshland, D. 2004. In vivo requirements for rDNA chromosome condensation reveal two cell-cycle-regulated pathways for mitotic chromosome folding. Genes Dev. 18: 76-87.

Huang, D. and Koshland, D. 2003. Chromosome integrity in Saccharomyces cerevisiae: the interplay of DNA replication initiation factors, elongation factors, and origins. Genes Dev. 17: 1741-1754.

Milutinovich, M. and Koshland, D.E. 2003. Molecular biology. SMC complexes—wrapped up in controversy. Science 300: 1101-1102.

Yu, H.G. and Koshland, D. 2003. Meiotic condensing is required for proper chromosome compaction, SC assembly, and resolution of recombination-dependent chromosome linkages. J. Cell Biol. 163: 937-947.

Lavoie, B., Hogan, E. and Koshland D.E. 2002. In vivo dissection of the chromosome condensation machinery: reversibility of condensation distinguishes contributions of condensin and cohesin. J. Cell Biol. 156: 805-815.

PloS Genetics in press. Lavoie, B.D., Tuffo, K.M., Oh, S., Koshland D. and Holm, C. 2000. Mitotic chromosome condensation requires Brn1p, the yeast homologue of barren. Mol. Biol. Cell 11: 1293-1304.

Koshland, D.E. and Guacci, V. 2000. Sister chromatid cohesion: the beginning of a long and beautiful relationship. Curr. Opin. Cell Biol. 12: 297-301.

Laloraya, S., Guacci, V. and Koshland, D. 2000. Chromosomal addresses of the cohesin-component, Mcd1p. J. Cell Biol. 151: 1047-1056.

Hartman, T., Stead, K., Koshland, D. and Guacci, V. 2000. Pds5p is an essential chromosomal protein required for both sister chromatid cohesion and condensation in Saccharomyces cerevisiae. J. Cell Biol. 151: 613-626.