Dissecting the role of MukF in the chromosome organisation by Escherichia coli MukBEF

Thesis

The organisation and segregation of chromosomes from all domains of life are mediated by the architecturally conserved Structural Maintenance of Chromosomes (SMC) proteins and their accessory factors. In Escherichia coli, the distantly related complex MukBEF retains the distinctive tripartite ring structure that has been proposed to topologically entrap DNA. Cyclical loading and unloading of DNA is regulated by ATP binding and hydrolysis, both of which are essential for SMC function. The kleisin MukF stimulates MukB ATPase activity. Uniquely, MukF forms stable dimers through the N-terminal Winged Helix Domain (WHD), thus potentially allowing for the formation of higher order complexes. Null mutations in any one of the mukBEF genes results in temperature sensitivity in rich media, disturbed organisation of the chromosome and anucleate cell production even under permissive conditions.

In this study a tripartite approach of biochemistry, genetics and in vivo imaging, provides insight into the functional importance of dimeric MukF. I have identified hydrophobic interactions between protomers that contribute to MukF dimerisation. In vitro assays show that full length MukF monomers are folded and stimulate MukB ATPase to wild type levels. However, in vivo, MukF dimerisation is essential for MukBEF function, as judged by the ΔMukBEF phenotype of temperature sensitivity in growth on rich media. Epifluorescence image analysis and Photoactivatable Localisation Microscopy (PALM) data indicate that chromosome interaction of MukBEF complexes formed with monomeric MukF is defective. Furthermore, an artificial dimerisation domain fails to rescue the ΔMukBEF phenotype. I propose that the formation of dimers of dimers is a prerequisite for the ability of MukBEF to transport chromosomal DNA by a “rope-climber” model and that the dimerisation domain may play a dynamic role in the coordination of MukBEF complexes.

Authors: Baker, R

• DOI: 10.5287/ora-vjok2oxod
• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford