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Process of infection with bacteriophage phi X 174 XXXVIII. Replication of phi chi 174 replicative form in vivo
Abstract:
The replication of bacteriophage phi X 174 replicative-form DNA has been studied by structural analysis of pulse-labeled replicative-intermediate molecules. Such intermediates were identified by pulse-labeling with [13H]thymidine and separated into four major fractions (A, B, C, and D) in a propidium diiodide-cesium chloride buoyand density gradient. Sedimentation analysis of each of these fractions suggests the following features of phi X replicative-form DNA replication in vivo. (i) At the end of one cycle of replication, one daughter replicative form (RFII) contains a nascent plus (+) strand of the unit viral length, and the other daughter RFII contains small fragments of nascent minus (-) strand. (ii) Asymmetry is also associated with production of the first supercoiled RFI after addition of pulse label in that only the minus strand becomes radioactive. (iii) A supercoiled DNA (RFI') seems to occur in vivo. This DNA is observed at a position of greater density in a propidium diiodide-cesium chloride buoyant density gradient than normal RFI. (iv) A novel DNA component is observed, at a density greater than RFI, which releases, in alkali, a plus strand longer (1.5 to 1.7 times) than the unit viral length. These results are discussed in terms of the possible sequence of events in phi X 174 replicative-form replication in vivo.
Insights
Bacteriophage phi X 174 DNA replication involves producing one daughter molecule with a full-length plus strand and another with minus strand fragments. A unique supercoiled DNA form and a novel component with extended plus strands were identified.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Bacteriophage phi X 174 utilizes a unique replication mechanism involving its replicative-form (RF) DNA.
- Understanding the in vivo replication process is crucial for deciphering viral genetic material dynamics.
Purpose of the Study:
- To structurally analyze pulse-labeled replicative-intermediate molecules of bacteriophage phi X 174 RF DNA.
- To elucidate the sequence of events during phi X 174 RF DNA replication in vivo.
Main Methods:
- Pulse-labeling of phi X 174 RF DNA with [3H]thymidine.
- Separation of replicative-intermediate molecules into four fractions (A, B, C, D) using propidium diiodide-cesium chloride buoyant density gradients.
- Sedimentation analysis of separated DNA fractions.
Main Results:
- One daughter RFII molecule contains a nascent unit-length plus strand, while the other contains nascent minus strand fragments.
- Asymmetric synthesis results in a radioactive minus strand during the production of the first supercoiled RFI.
- Identification of a denser supercoiled DNA (RFI') and a novel DNA component releasing plus strands 1.5-1.7 times the unit viral length.
Conclusions:
- The study reveals key features of phi X 174 RF DNA replication, including strand asymmetry and the existence of novel DNA forms.
- These findings provide insights into the intricate steps of viral DNA replication in vivo.
- The identified novel DNA component suggests a unique mechanism for plus strand elongation during replication.