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Title: Investigation of the DNA binding domain of nuclear factor I
Author: Freeman, Alasdair D. J.
Awarding Body: University of St Andrews
Current Institution: University of St Andrews
Date of Award: 1999
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Nuclear Factor I is a cellular transcription factor involved in the initiation of adenovirus type 2 replication. It exists as a family of proteins and comprises two distinct domains. The N-terminal domain is highly conserved between species and is involved in DNA binding, dimerisation, and stimulates adenovirus replication. The C terminal domain is responsible for the transcriptional activation. In this work, the N terminal, DNA binding domain of Nuclear Factor I, was cloned in two different expression systems. Using recombinant baculovirus, the unmodified protein and a GST fusion product were expressed in insect cells. Although both proteins were expressed at high levels, it was impossible to purify the fusion protein, while after purification, the unmodified protein remained heterogeneous. Expression in E. coli using the expression vector pET22b and pGEX2T produced inclusion bodies. The insoluble material was extracted, solubilised using guanidine HCl, then folded in vitro using new additives known as non-detergent sulphobetaines. In vitro folding was optimised and yields of up to 8% could be obtained. The affinity of the refolded material for a specific DNA oligonucleotide was determined in a gel electrophoresis DNA binding assay and was identical to the native protein purified from baculovirus infected insect cells. The structure of the Nuclear Factor I DNA Binding Domain was investigated using limited proteolysis followed by separation on SDS PAGE, N terminal sequencing and mass spectrometry. Residues 1 to 165 formed a compact domain. Residues 166 - 181 were extremely sensitive to degradation in the absence of DNA but fully protected in the presence of specific DNA. The C terminal region from residue 182 could be degraded in both conditions. Along with two other regions previously determined which bind DNA, the region between residues 166 and 181 is required for DNA binding. Differential labelling using iodoacetate showed that cysteine residues 95 and 111 were modified in the free protein which resulted in an inactive protein but were protected in the presence of DNA. This demonstrates their direct involvement in DNA binding.
Supervisor: Hay, Ronald Thomas Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QH465.F8 ; Radiogenetics