Replication of bacteriophage M13: XI. Localization of the origin for M13 single-strand synthesis

Abstract

The origin for bacteriophage M13 single-strand synthesis has been localized by two different experimental approaches: (1) localization of the discontinuity in late life-cycle RFII ‡ , one of the initial product molecules during single-strand synthesis, and (2) determination of a gradient of radioactive label in RFI pulselabeled during single-strand synthesis. M13 RFII formed during single-strand synthesis contains a single discontinuity in the viral strand. Cleavage of this RFII species with the HindII restriction enzyme yields two discrete viral strand fragments that locate the discontinuity at a distance approximately 10% of the genome from the HindII restriction site. RFII molecules were also used as template for limited repair synthesis with Escherichia coli DNA polymerase I and 32P-labeled deoxynucleoside triphosphates. Upon treatment of these repaired RFII with HpaII restriction endonuclease and electrophoresis in an agarose gel, the discontinuity was localized in a specific restriction fragment, the F fragment. The existence of a gradient of pulse label in RFI isolated late in infection was determined by mixing [3H]thymidine pulselabeled RFI and uniformly-labeled [32P]RFI, treating this mixture with HpaII endonuclease, separating the fragments by electrophoresis in an agarose gel, and examining the ratio of pulse label to uniform label in the restriction fragments. The ratio varied with the position of the fragment on the cleavage map indicating the temporal order of synthesis. Both experimental approaches place the origin of M13 viral-strand synthesis within the HpaII fragment F. This fragment is approximately 400 base-pairs in length and is within an intergenic space between genes II and IV on the genetic map. This region was previously shown to contain the origin for complementary-strand synthesis in vitro.