Identification, assembly and characterization of the condensin complex from Saccharomyces cerevisiae;
The focus of this thesis was to study the budding yeast “Condensin” complex and mechanism(s) by which this complex directs chromatin reconfiguration. The protein components of the S. cerevisiae condensin were positively identified using a combination of affinity isolation by TAP pull-down and MALDI-MS. Unambiguous assignment of the theoretical trypsin fragment fingerprints of five ORF's: SMC2, SMC4, YCS4, YCG1 & BRN1 were made. These ORF's were cloned for over-expression in yeast and each protein purified in order to define their biophysical and functional properties. Most importantly, the holo-condensin (2/4/4/1/1) complex and two condensin subcomplexes composed either of Smc2p and Smc4p (2/4), or Ycs4p, Ycg1p and Brn1p (4/1/1) have been reconstituted. The Smc2/4 condensin induces chiral knot formation but does not introduce appreciable positive supercoiling (writhe) into closed circular DNA substrates. Neither Smc2p nor Smc4p hydrolyzes ATP, but the Smc2/4 complex formed upon mixing of the two purified proteins does, thus proving that this SMC heterodimer is an ATPase. Futhermore, site directed smc2 / 4 mutants, which can not hydrolyze ATP, induce chiral knot formation, suggesting that the knotting activity: (1) is not energy dependent, (2) does not require cell cycle dependent modifications, and (3) does not require the participation of the non-SMC subunits. The fact that the 2/4 subcomplex does not appear to introduce appreciable writhe when bound in a knotting proficient manner runs counter to the currently proposed mechanism of chiral knot formation, and thus a potential solution to an intriguing topological problem is presented.
University of Utah;
Mitotic Regulation; Proteins;
Saccharomyces cerevisiae; Chromosomes;
University of Utah;
Relation-Is Version Of
Digital reproduction of “Identification, assembly and characterization of the condensin complex from Saccharomyces cerevisiae.” Spencer S. Eccles Health Sciences Library. Print version of “Identification, assembly and characterization of the condensin complex from Saccharomyces cerevisiae.” available at J. Willard Marriott Library Special Collection. QK3.5 2003 .S73.