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Nature of phi chi 174 linear DNA from a DNA ligase-defective host
Abstract:
Linear DNAs have been prepared from phiX phage and from phiX RF II (double-stranded circular form of phiX DNA, formed during infection and nicked in one or both strands) molecules derived from infection at the restrictive temperature of Escherichia coli ts7, a host mutant with a temperature-sensitive DNA ligase activity. The linear DNA from these phages can be circularized by annealing with fragments of phiX RF DNA produced by the Haemophilus influenzae restriction nuclease. The circularization experiment indicated that the site of breakage of the linear phage DNAs is not unique nor confined to a particular region of the genome. These linear DNAs were less than 0.1% as infective as circular phage DNA. The linear, positive strand of late RF II DNA, however, is uniquely nicked in the region of the phiX genome corresponding to cistron A. Although a low level of infectivity is associated with the linear DNA derived from late RF II, this infectivity appears to be a result of the association of linear positive and linear negative strands during the infectivity assay.
Insights
Linear phage DNA molecules were prepared and found to be poorly infectious. Circularization experiments revealed that DNA breakage sites are not unique, though specific nicks occur in certain DNA forms.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Bacteriophage phiX DNA replication involves circular intermediates (RF II).
- Escherichia coli mutants with temperature-sensitive DNA ligase can affect DNA processing.
- Understanding DNA breakage and repair is crucial for viral replication and host-pathogen interactions.
Purpose of the Study:
- To characterize linear DNA forms derived from bacteriophage phiX.
- To investigate the infectivity and breakage sites of these linear DNA molecules.
- To explore the role of host DNA ligase in DNA integrity.
Main Methods:
- Preparation of linear DNA from phiX phage and RF II molecules.
- Infection of a temperature-sensitive E. coli mutant (ts7) at restrictive temperatures.
- Circularization of linear DNA using fragments of phiX RF DNA generated by restriction nucleases.
- Assessment of DNA infectivity through comparative assays.
Main Results:
- Linear phiX DNAs exhibit significantly reduced infectivity (<0.1%) compared to circular forms.
- Circularization experiments demonstrate that breakage sites in linear phiX DNA are not unique.
- A specific nicking site was identified on the linear, positive strand of late RF II DNA, localized to cistron A.
- The low infectivity of late RF II-derived linear DNA is attributed to the association of both positive and negative strands.
Conclusions:
- Linearization of phiX DNA results in a substantial loss of infectivity.
- The lack of a unique breakage site suggests diverse mechanisms for DNA strand breakage in phiX phage.
- Specific nicking in cistron A of late RF II DNA indicates potential regulatory roles or replication intermediates.
- The infectivity assay highlights the importance of intact circular DNA and strand association for successful phage infection.