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Abstract

During cell division, the genetic material of each cell, the DNA undergoes the most drastic rearrangements. The amorphous interphase chromatin condenses into short discernable mitotic chromosome during this process governed by a protein complex known as condensin. Condensin is a member of the structural maintenance of chromosome (SMC) family and comprised of five subunits. SMC2 and SMC4 along with a kleisin subunit, Brn1 form a tripartite ring and association of two additional HEAT (huntingtin, elongation factor 3, protein phosphatase 2A, Tor1 kinase) repeat proteins, Ycg1 and Ycs4 makes the functional pentameric complex. It has been postulated that condensin compacts genomes by a process called ‘loop extrusion’ where DNA is reeled in into a loop, but it wasn’t until recently that this was shown in real time. Purified condensin complex from budding yeast has been shown to extrude large DNA loops in vitro. On this account, the aim of my Ph.D. thesis was to generalize this process of ‘loop extrusion’ and get mechanistic insights for this process. I first purified condensin complex of a thermophilic fungus called Chaetomium thermophilum (Ct), and characterized it biochemically and biophysically. I showed that the Ct condensin is intact and that it can compact DNA. Additionally, it has DNA stimulated ATPase activity and can readily bind DNA. Using advanced microfluidics and super-resolution microscopy, I then showed that the Ct condensin complex extrudes DNA loops in an asymmetric manner. To my surprise, a condensin complex lacking the Ycg1 subunit, previously attributed as the anchor module necessary for stable DNA binding, still showed DNA compaction, DNA-stimulated ATPase activity and even a DNA binding affinity similar to wildtype complex. My imaging experiments showed this sub-complex also loop extrudes, however unlike wildtype complex frequently changing direction. It is only when I in addition to Ycg1 also deleted the so-called Brn1 ‘safety belt’ (Brn1515-637), I no longer observed loop extrusion. Finally, I was able to show that patches of positive and of hydrophobic residues at the amino-terminal end of the ‘safety belt’ are essential for loop extrusion. Purely asymmetric loop extrusion has been questioned on its ability to fully compact large eukaryotic genomes and loop extrusion with frequent direction changes has so far only been proposed in theoretical studies as a means to overcome this limitation. My discovery of direction changes during loop extrusion provides the first real time experimental proof that such extrusion exists and that a mixture of asymmetric extrusion with direction change can accomplish full genome compaction. It is tempting to speculate that the mode of action shown for a thermophilic condensin protein complex could serve as example for the universal mode of chromosome compaction.