| This dissertation reports a mechanical study of a bacterial condensin protein complex and provides the evidence that condensin is able to compact DNA in an ATP-dependent manner. The model system under study is the condensin complex MukBEF, which is responsible for proper chromosome organization and segregation in Escherichia coli. At the center of MukBEF lies a pair of SMC proteins (S&barbelow;tructure M&barbelow;aintenance of C&barbelow;hromosome), members of which are responsible for a wide variety of chromosome dynamics including chromosome condensation. Using atomic force microscopy (AFM), I observed the head-tail architecture of MukBEF demonstrating that this condensin adopts a closed rod configuration with the two non-SMC subunits bound near the head region. When bound to DNA, MukBEF introduces ∼110° bends into DNA segments in an ATP-independent manner.; Using a fluorescence-based assay, preliminary results demonstrate ATP-dependent DNA condensation activity in the presence of MukBEF. MukBEF-induced DNA condensation is initiated from one or two nucleation sites on DNA, indicative of high degrees of cooperativity. The average rate of condensation is 256 nm per minute.; When single molecules of DNA are subjected to stretching in the presence of MukBEF using laser tweezers, the persistence length of DNA reduces from 45nm to 25nm in an ATP-independent manner, confirming that MukBEF bends DNA independent of ATP. During the course of investigation we observed DNA unzipping by mechanical forces giving rise to a force sawtooth pattern at ∼17pN. Average free energy associated with force-induced DNA melting for one basepair is estimated to be ∼2.4kcal/mol.; Using magnetic tweezers to investigate how MukBEF modifies DNA elastic responses in the low force regime, I observed MukBEF-induced ATP-dependent DNA condensation against forces <0.1 pN, leading to the formation of a compact rigid fiber structure. The condensation-decondensation transition occurs at ∼1 pN. Based on these observations, I propose a molecular model about how MukBEF induces DNA condensation in vitro and in vivo. In this model, MukBEF binds and bends DNA in a nucleotide-independent manner. ATP-mediated intermolecular interactions between DNA-bound MukBEF promote the formation of a compact rigid nucleoprotein filament with DNA loops spanned between MukBEF molecules. Because of the relatively weak interactions, the filament structure is likely to be dynamic in vivo. Therefore MukBEF functions as a higher order chromosome organizer indispensable for proper chromosome segregation. |
