The phi-XA protein plays a role in the site specific cleavage of the (+) strand of phi-X RFI DNA, the displacement reaction, a ligation reaction and a transfer reaction. These reactions require a 30 nucleotide conserved sequence found in all icosahedral phages. Alterations of this region at the 3'-end prevent binding, alterations around the cut site control endonuclease action and alteration at the 5'-end prevent the transfer reaction. Two tyrosine moieties in the phi-XZ protein can form a covalent linkage with the dAMP residue at nucleotide 4306; these polypeptides will be isolated and sequenced. The sites at which the phi-XA protein interacts with the conserved 30 nucleotide sequence will be determined by methylation protection and by DNase """"""""footprinting"""""""" experiments. RFI plasmids containing one conserved sequence and one altered sequence on the same strand will be constructed. The above reactions will be examined. The influence of phi-X174 prohead and the phi-XC and J proteins on the phi-XA catalyzed reactions will be studied. The role played by each protein involved in the in vitro replication of Ad DNA will be examined. The initiation reaction (pTP-dCMP complex) requires three Ad-encoded proteins, the pTP, the Ad pol and the Ad DBP plus one host protein nuclear factor I. This reaction requires two sequences present within the first 50 bp at each end of Ad DNA. One sequence lies between nucleotides 9-18, while the other is the factor I binding site (18-49). DNA synthesis will be studied in mutants in which the position of these two sites and their polarity will be varied. We plan to clone these proteins; monoclonal antibodies to nuclear factor I have been isolated and we plan to isolate the Ad Polpolymerase and the pTP protein by linking their cDNA to expression vectors. Replication of the displaced strand will be examined. Plasmids containing ori regions that are terminally redundant yield single-strands that form panhandle structures which are replicated. The replication of SV-40 DNA has been carried out in vitro. HeLa cell extracts plus SV-40 large T antigen support replication of plasmids containing the SV-40 origin sequence; no replication was observed with plasmids lacking the origin site. Intermediates in the reaction accumulate and migrate as complicated structures. These intermediates arise from the ori region and move in both directions. We are now in the process of purifying the proteins involved in the replication of SV-40 DNA.
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