Origin and direction of φX174 double- and single-stranded DNA synthesis

Abstract

The origin and direction of both φX174 double-stranded and single-stranded DNA synthesis has been determined by pulsing replicating viral DNA molecules with [ 3H]thymidine for periods of less than one round of DNA synthesis and examining distribution of activity in the Haemophilus influenzae restriction endonuclease (Hin) DNA fragments of these molecules. In early RFI and RFII DNA intermediates in double-stranded DNA replication, gradients of label were observed which started in the R3 fragment (cistron A) and increased towards the R4 fragment (cistron H). The origin of synthesis is near the R4/R3 junction of the R3 fragment. Thus, φX174 double-stranded DNA synthesis proceeds clockwise around the genetic map (5′ → 3′), in one direction only and starting in the region of cistron A, a conclusion consistent with the genetic experiments of Baas & Jansz (1972). Similar experiments with the gapped late RFII DNA molecules that have just completed a round of single-stranded viral DNA synthesis demonstrated that φX174 single-stranded DNA synthesis also has a single origin of replication in the region of cistron A, and that the synthesis moves in the 5′ → 3′ direction, around the genetic map. The gap in both the early and the late RFII DNA molecules also appears to be in the R3 fragment containing cistron A.