Nuclear architecture and nuclear function appear to go hand in hand: defects in nuclear organization are associated with aging and diseases such as cancer. In a previous study (Campbell et al, 2006) we found that in yeast nuclear shape is determined, in part, by the chromatin, which appears to have the ability to self organize. In this study our goal is to identify proteins that contribute to the global organization of chromatin. Our approach is based on the assumption that chromatin movement is restricted by proteins that contribute to global chromatin organization, and that inactivating these proteins will increase chromosome mobility. ? ? To examine whether chromosome movement is restricted, we are developing a recombination-based assay in which we can quantify the repair of a double stand break in a target gene via recombination with an ectopic donor gene. The specific assay we are using involves a site-specific endonuclease mediated double-strand break at the target site. The donor site has homology to the broken site. This affords the cell a chance to repair the break through homologous recombination (HR). We are also flanking the break site with homologous sequences, thereby providing the cell with another avenue to repair the break. Specifically, exonuclease mediated degradation of the 5 ends at the break will, when it has reached the homologous sequences flanking the break site, allow single-strand annealing (SSA) to seal the break. By using the flanking homologous sequences we are defining a time window during which HR can take place, before the sequences involved in SSA are exposed. These two competing reactions should account for most of the repair of the breaks since non-homologous ending joining is inefficient in yeast. Fewer constraints on the movement of these sites will lead to more frequent collisions between ectopic sites, favoring repair of the break by HR at the expense of SSA. Incorporation of appropriate genetic markers along with molecular analysis will allow us to determine the ratio of these two events. Initially, different chromosomal and non-chromosomal contexts (e.g. plasmids or small looped out DNA segments) will be compared. If we find that chromosome movement is indeed constraint, we will use the recombination assay to look for mutations that increase chromosome movement. We expect that some of these mutations will lie in genes involved in global chromatin organization.
| Cohen-Fix, Orna (2010) Cell biology: Import and nuclear size. Nature 468:513-6 |
| Webster, Micah; Witkin, Keren L; Cohen-Fix, Orna (2009) Sizing up the nucleus: nuclear shape, size and nuclear-envelope assembly. J Cell Sci 122:1477-86 |
