Structural and functional studies of the Dsc1 cell cycle transcription factor complex in fission yeast

Dunlop, Allan John (2004) Structural and functional studies of the Dsc1 cell cycle transcription factor complex in fission yeast. PhD thesis, University of Glasgow.

Abstract

The fission yeast DSC 1 (DNA synthesis control) transcription factor complex regulates cell cycle-specific periodic transcription of a group of genes at the G1-S phase transition during the mitotic cell cycle, by binding to MCB (Mlul cell cycle box) sequence elements common to their promoters. Included in this group are several genes whose functions are required for the onset and progression of S phase, such as cdc22+ (the large subunit of ribonucleotide reductase), cig2+ (the major S phase cyclin) and the DNA replication licensing factors cdc18+ and cdt1+. In concert with cyclin-dependent kinase activity, DSCl function is required for passage of START and entry into the mitotic cell division cycle. Similar gene expression programmes exist in both budding yeast and humans controlled by the SBF/MBF and E2F transcription factors, respectively. Fission yeast DSC1 comprises two related DNA-binding subunits, Res1p and Res2p, each bound to a single molecule of the regulatory Cdc10p protein. In addition, the Rep2p protein has a transcriptional activator function (replaced by Rep1p in the meiotic cycle). Knowledge about functional aspects of each of the DSC1 components has been greatly enhanced by genetic and biochemical studies. However, to date, these proteins remain poorly characterised at the atomic level, with little known about structure beyond their amino acid sequence. The aim of this study was to clone and bacterially express the individual DSC1 genes, to provide sufficient protein to carry out more detailed biophysical and functional studies. The cdc10+, res1+, res2+ and rep2+ genes, together with the putative meiotic subunit rep1+, were cloned and overexpressed in E. coli as N-terminal histidine-tagged fusion proteins. Inclusion of the His-tag facilitated purification of the proteins by affinity chromatography. Each recombinant protein (with the exception of His-Cdc10p) was shown to function in vivo-, ectopic expression of His-resl+, His-res2+, His-rep1+ or His-rep2+ rescued the cold-sensitive lethality of the fission yeast Deltares1 mutant strain. Bacterially expressed His-Res1p, His-Res2p and His-Rep2p were recovered in soluble form, whilst His-Rep1p and His-Cdc10p were detergent-solubilised from inclusion bodies. His Res2p was expressed and purified in yields sufficient to undertake biophysical analyses. Both His-Rep1p and His-Cdc10p were solubilised and purified from inclusion bodies in yields sufficient for structural studies, although initial biophysical data suggests that re-folding strategies will be required to obtain active preparations of these proteins. In electrophoretic mobility shift assay experiments, neither His-Res1p nor His-Res2p displayed detectable MCB-specific DNA-binding in vitro. Intriguingly, replacement of the His- tag with an N terminal GST-tag conferred detectable MCB-specific DNA-binding upon both proteins. These results suggest that efficient DNA-binding requires dimerisation, a property that, at least in vitro, is apparently not naturally intrinsic to either Resp protein. Taken together, the results presented in this study provide a significant basis with which to undertake future structural analyses of these proteins. The implications of these results for further in vitro studies are discussed.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Cellular biology.
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: McInerny, Dr. Chris and Lindsay, Prof. Gordon
• Date of Award: 2004
• Depositing User: Enlighten Team
• Unique ID: glathesis:2004-71137
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 10 May 2019 10:49
• Last Modified: 15 Jun 2021 10:36
• URI: https://theses.gla.ac.uk/id/eprint/71137