The mechanism of replication of øX174 DNA: XII. Non-random location of gaps in nascent øX174 RF II DNA

Abstract

Nascent oX174 replicative form (RF) DNA purified from phage oX-infected Escherichia coli was labelled in a “gap-filling” reaction with [α-32P]dNTPs using phage T4 DNA polymerase and DNA ligase. Three different methods of analysis indicated that the gaps were not randomly located in either the viral or the complementary strand. o (1) RF DNA labelled with 32P in the gaps was treated simultaneously with the restriction endonucleases from Hemophilus influenzae and Hemophilus aegyptius and the resulting fragments separated by electrophoresis on agarosepolyacrylamide gels. The distribution of the 32P in the fragments of the gaplabelled RF was compared to the distribution of 32P present in the fragments of uniformly-labelled oX RF DNA and found to be different. (2) The viral and complementary strands of the gap-labelled RF were separated by reannealing in the presence of excess viral strands, followed by hydroxyapatite chromatography; the separated strands were then digested with formic acid/diphenylamine. The distribution of pyrimidine oligonucleotides in each strand was determined by ionophoresis-homochromatography and found to differ from the distribution observed when the entire strand was uniformly labelled. (3) The number of gaps in the population of RF molecules under study was determined from the amount of 32P acquired from [γ-32P]ATP in a reaction catalyzed by polynucleotide kinase. In separate reactions the amount of each of the four deoxynucleoside monophosphates incorporated in the gap-filling reaction was determined; combination of this information with the number of gaps indicated that the average gap in both the viral and the complementary strands was 13 to 16 nucleotides long. The proportions of the four nucleotides incorporated into the gaps in the viral and complementary strands were different from the proportions found in the complete viral and complementary strands, and were biased in favour of the pyrimidine nucleotides in both cases. These observations suggest that synthesis of all viral DNA during oX RF replication is initiated at non-randomly distributed sites on the oX genome. The origin of replication appears to be in the Z6b Hemophilus aegyptius restriction endonuclease cleavage fragment and to contain the unique C6T pyrimidine tract.