Deducing the In Vivo Supercoiling Capabilities of the Condensin II Complex in Drosophila Melanogaster

Abstract

Condensins are some of the most ancient and well conserved protein complexes. Although much work has focused on the role of condensins in chromosome condensation during mitosis, other unique functions are less characterized. We have previously shown that condensin II functions in a novel mechanism to unpair interphase homologs in Drosophila melanogaster. We hypothesize that condensin II mediates this unpairing by inducing supercoils, which promote chromatid separation. Condensins have already been shown to induce positive supercoils in vitro, but the aim of this study was to develop an assay to test in vivo supercoiling by the condensin II complex. By measuring the distance between two fluorescently labeled loci along a chromosome, we have shown longitudinal axial shortening when condensin II subunits are expressed. We also show that ethidium bromide staining, which binds preferentially to negatively supercoiled DNA, increases in both diploid and polyploid larval tissue when Cap-H2, a condensin II subunit, is overexpressed. Finally, we are currently working on assaying circular DNA constructs in both larvae and Drosophila cell culture to look for changes in superhelical density upon overexpression of Cap-H2.