RESEARCH INTERESTS
Chromosome Structure and Segregation
Summary: Through the analysis of factors that mediate chromosome condensation and sister chromatid cohesion during mitosis and meiosis, Douglas Koshland hopes to elucidate the underlying molecular mechanism of chromosome structure and the principles that govern chromosome evolution.
During mitosis, the spindle, a complex microtubule-based machine, plays a critical role in the segregation of replicated chromosomes (sister chromatids). Sister chromatids are not, however, passive substrates in this process but rather actively participate in their own segregation by virtue of three specific structural features. First, each sister chromatid has a centromere, a region that mediates the attachment and movement of chromosomes on the microtubules of the spindle. Second, sister chromatids are paired. 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. 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.
The third structural feature of sister chromatids is their condensation, which helps to minimize the entanglement of chromosomes while they move during mitosis. It 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.
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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 ~13-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 and an extensive coiled-coil domain. 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 elucidate their molecular functions, we have initiated a genetic approach to trap intermediates in Smc complex function and assembly.
Condensins and cohesins provide valuable tools to analyze the mechanism of mitotic chromosome folding. This process is likely to occur through specific folds (e.g., helices and loops) analogous to folding of proteins by the formation of a helices and b sheets. Our in vivo analysis has shown that condensins are required for both the establishment and maintenance of condensation. We have identified two intermediates in the folding process, a gathering step and a resolution step. One idea is that the initial gathering may reflect a particular fold generated by condensin binding, while the resolution might represent higher-order organization of condensins after binding. The function of condensins in chromosome folding will not be understood until we have assays to identify and characterize chromosome folds. For this purpose we are exploiting the 10-kb tandem repeats of the budding yeast rDNA locus, the in vitro condensation activity of Xenopus extracts, and electron microscopy to provide a new readout for chromosome folding.
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. The proper repair of broken chromosomes is essential to prevent the formation of gross chromosomal rearrangements (GCRs). We developed a simple and rapid assay for GCRs, exploiting yeast artificial chromosomes (YACs) in Saccharomyces cerevisiae. With this assay we performed the first genome-wide screen for proteins that prevent GCRs. These analyses identified condensins as well as novel factors in DNA replication and elongation. Our results also provide insights into how the interplay of the DNA replication factors has constrained the evolution of chromosome replication. In an independent study we show that cohesins also play an important role in chromosome integrity. Many cohesin molecules are recruited to the site of a double-strand break, covering as much as one-third of the chromosomes. These cohesins use the sister chromatid as a template to stimulate repair. How this macrodomain of cohesin stimulates repair is the focus of our current work.
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.
Because of the ability to manipulate genome structure and function, the study of chromosome structure in yeast hybrids will allow us to test a number of possible contributions to chromosome evolution, such as the contribution of disperse repetitive DNA, whole-genome duplication, sequence divergence, and protein buffering.
