Bacteriophage φx174 DNA synthesis in a replication-deficient host: Determination of the origin of φX DNA replication

Abstract

We have reinvestigated bacteriophage φX DNA synthesis in rep 3 − cells. In these cells adsorption, transcription and translation of φX occurs normally, but φX replicative form DNA replication is blocked. After infection, φX DNA was isolated and separated by propidium diiodide/CsCl gradients into RFI † † Abbreviations used: RFI, replicative form DNA with both strands closed; RFII, replicative form DNA with one or more discontinuities in either strand; CAM, chloramphenicol; m.o.i., multiplicity of infection; PrI 2, propidium diiodide. HaeIII and HindII, restriction endonucleases from Haemophilus aegyptius and Haemophilus influenzae Rd, respectively. Z and R, DNA fragments produced by HaeIII and HindII, respectively. and RFII, followed by chromatography on BND-cellulose. In addition to the results of Francke & Ray (1971, 1972), we found an increasing amount of viral strand DNA synthesis in each fraction as time of infection proceeds. The occurrence of viral strand DNA synthesis is dependent on the presence of a functional cistron A product. In the presence of 150 μg chloramphenicol/ml or in infections with am18 (gene A) no viral strand DNA synthesis could be detected. After completion of the parental RF molecule, [ 3H]thymidine enters almost exclusively into viral strand DNA. The greater part of this viral strand DNA is found in genome-length φX. DNA in both RFI and RFII. Alkaline sucrose sedimentation of the RFII indicates that post-infection viral DNA label is only in linear φX DNA. These results strongly suggest that an extensive nick translation of the viral strand of RFII, followed occasionally by a conversion to RFI, takes place after synthesis of a functional cistron A product. This conclusion could be confirmed by restriction enzyme analysis of φX RF DNA, isolated from rep 3 − cells infected with 32P-labeled phage in the presence of [ 3H]thymidine. With the restriction enzyme HaeIII a gradient in the 3H 32P ratio of the fragments was obtained extrapolating into the fragment Z 6B. From this gradient the origin of the φX DNA nick translation could be located in the left half of Z 6B at approximately 65% (185 base-pairs) from the Z 6A-Z 6B junction.