| Summary: | The DNA sequences of many small staphylococcal plasmids possess open reading frames with 70-80 sequence identity to that of the repD product of the chloramphenico1-resistance plasmid pC221. This thesis describes the results of studies of the REP D protein, required for initiation of replication of pC221 both in vivo and in vitro. REP D was expressed in Escherichia coli, using a purpose-built expression vector, as 10-15% of the total cellular protein. Purification yields 40-50mg of REP D from one litre of induced culture. The protein displays a molecular weight of 35 kDa on denaturing gel electrophoresis, in close agreement with that predicted from the DNA sequence, and 75 kDa by gel filtration of the native form, suggesting a dimeric quaternary structure. N-terminal amino acid sequence analysis of REP D revealed the absence of the presumed initiator methionine residue. In vitro REP D has both sequence-specific endonuclease and type-I topo-isomerase activities. The target is oriD, the replication origin of pC221, contained within the repD reading frame. Supercoiled pC221 and plasmids with related origin sequences (such as pT181) are relaxed by REP D in high ionic strength buffers, whereas relaxed, covalently closed plasmid pC221 only is nicked by REP D at a precise location in the (+) strand (oriD) under conditions of low ionic strength. Both reactions require Mg2+ but not ATP. Binding of REP D to oriD was demonstrated, using restriction fragments of pC221 and synthetic oligonucleotides. Competition experiments suggest two DNA binding sites. The first is involved in the recognition of the conserved cleavage site. The second site discriminates between such potential origins by binding only to a "specificity" sequence present within oriD. After nicking the relaxed origin DNA substrate REP D is found to be covalently attached to the resultant 5' end. Radiolabelling and hydrolysis revealed the linkage to be via a phosphodiester bond to a tyrosine residue. Peptide sequencing identified the tyrosine to be Tyr188 of REP D, conserved in the sequences of all six related REP proteins known. A mechanism for initiation of plasmid replication is suggested on the basis of these results. The nicking reaction is thought to trigger the initiation of replication of pC221, the free 3' hydroxyl group generated priming synthesis of the new (+) strand. The requirement in vitro for a relaxed substrate to demonstrate the absolute sequence specificity seen in vivo suggests the local topology of the origin region may have low superhelical density in vivo.
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