Ali-Khan, Nadeem; (2002) Biochemical characterisation of the eukaryotic cell cycle regulatory proteins, E2F and pRB. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Control of the cell cycle is partly mediated by a transcriptional regulatory mechanism whose components include the pRb family of tumour suppressors (pRb, p130, p107) and the E2F/DP heterodimeric transcription factors. Each of these heterodimers consists of one member of the E2F family of proteins (E2Fs 1- 6) and one of the DP family (DPs 1 and 2). E2F/DP activation of cell cycle genes is negatively regulated by cyclin A-CDK2-mediated phosphorylation of DP. The formation of a complex between E2F/DP and a pRb family protein leads to antiproliferative transcriptional repression. Binding of the Human Papillomavirus (HPV) E7 oncoprotein to pRb blocks the interaction of the tumour suppressor with E2F/DP as part of a viral cell transformation mechanism. Fragments of pRb, E2F-1 and DP-1 were over-expressed and purified by chromatographic means prior to their biochemical characterisation. Using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, attempts were made to determine high affinity DNA-binding sites for E2F homodimers. Although such sites were not identified, important considerations relating to the SELEX protocol are highlighted by these experiments. Electrophoretic Mobility Shift Assays were used to demonstrate that the interaction of fragments of E2F-1 and DP-1 with their cognate DNA could be inhibited by phosphorylation by cyclin A-CDK2. Furthermore, mutation of one of two putative phosphorylation sites in DP-1 resulted in a reduced rate of cyclin A- CDK2-dependent loss of DNA binding. Isothermal titration calorimetry studies revealed that not only does pRb interact with the minimal pRb-binding region of E2F-1, but also with additional regions outside of the transactivation domain. I present data showing that HPV E7 competes for binding to pRb with constructs of E2F-1 incorporating these additional regions. Our results suggest that the CR3 domain of E7 competes with the marked box region of E2F-1 for binding to pRb.

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