The replication of bacteriophage ФX174 DNA in vitro: Temperature effects on repair synthesis and displacement synthesis

Abstract

1. The effect of incubation temperature on the extent of in vitro viral DNA synthesis by DNA polymerase and the kinds of products synthesized has been investigated. 2. At temperatures below 22–25° in vitro DNA synthesis by DNA polymerase, using circular ФX174 DNA as a template, is limited to one cycle of replication. All circular phage DNA can be converted to completely double-stranded molecules; then net polymerization ceases. The number of initiations per template is shown to vary with changes in the relative rates of initiation and polymerization. 3. Most segments of the nicked complementary strand, formed at high levels of initiator, can be joined by polynucleotide-joining enzyme. The lstar nick is less frequently closed at 15°. This suggests a unique problem associated with closure of the lstar nick to form a complete circular DNA strand. 4. An apparent temperature threshold exists for greater than 1-fold synthesis. At temperatures above this threshold, product strands are elongated to several phage DNA unit lengths. The existence of the apparent temperature threshold suggests that a cooperative structural change may be necessary to allow the initiation and/or propagation of the synthesis leading to extended strands. 5. The activation energy measured for the continuation of synthesis beyond 1-fold is 39±4 kcal/mole. That for the first cycle of replication is 17±2 kcal/mole. It is suggested that denaturation of a small number of base pairs (2–4) in the region of the growing point is needed to initiate and sustain greater than 1-fold synthesis.