Bacteriophage phiX174 RF DNA replication in vivo: a biochemical study.

Abstract

Abstract We have studied the replication of bacteriophage φX174 replicative form (RF) DNA by biochemical analysis of pulse-labelled replicative intermediates and covalently closed RFI † DNA. The pulse-labelled DNA was isolated and separated into fractions which were shown to contain rolling circles, partially double-stranded molecules, RFI and RFII molecules, respectively. In these molecules the position of the pulse label and its strand specificity was determined. The results obtained were strongly dependent on the method used to stop the pulse-labelling. When the pulse is ended by poison (KCN and NaN 3 ) and fast cooling significantly more label is found in the viral than in the complementary strand. In agreement with the rolling circle model, the majority of the viral strand label is present in linear genome length and longer than genome length φX DNA molecules. The radioactivity in the RFII fraction is found almost exclusively in linear viral φX DNA of genome length. Restriction enzyme analysis of this fraction indicates that the φX viral strand DNA synthesis terminates in the Hae III restriction fragment Z 6B . In contrast, the main part of the labelled complementary strand φX DNA is found in short pieces. Full length, pulse-labelled complementary strands are present in the RFI fraction and to a minor extent in the RFII fraction. Restriction enzyme analysis of full length complementary strands, from RFI and RFII, shows that there is no unique termination site for the complementary strand DNA synthesis. Analysis of the pulse-labelled covalently closed RFI fraction indicates that this fraction contains a collection of molecules with increasing superhelix density, ranging from relaxed RFI up to fully super-twisted RFI DNA. This suggests that at the end of a replication cycle the conversion of RFII into superhelical RFI DNA involves the formation of relaxed RFI DNA and the successive introduction of superhelical turns. When the pulse is ended by pouring the infected culture into a phenol/ethanol solution less radioactivity is found to be incorporated into φX DNA. Furthermore, the distribution of the label between the complementary and viral strand is more equal, and significantly more radioactivity is found in short pieces. A comparison of both stopping methods indicates that fast cooling in poison does not stop efficiently φX. RF DNA replication, especially the viral strand DNA synthesis and DNA ligase action.