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Interaction of the bacteriophage phi 29 protein p6 with double-stranded DNA
I Prieto1, M Serrano, J M Lázaro
1Centro de Biología Molecular, Universidad Autónoma de Madrid, Canto Blanco, Spain.
Abstract:
The Bacillus subtilis bacteriophage phi 29 protein p6 binds to double-stranded DNA, but not to single-stranded DNA, as determined by a gel retardation assay. The nature of the interaction was further studied by DNase I "footprinting" experiments. Protein p6 binds to fragments containing the right or left terminal sequences of phi 29 DNA, producing a characteristic pattern of hypersensitive bands spaced about 24 nucleotides apart along most of the fragment, flanking protected regions. Binding of protein p6 to an internal phi 29 DNA fragment was also observed, but the footprint pattern was more salt sensitive than that obtained with the terminal phi 29 DNA fragments. By electron microscopy, protein p6 was shown to cover the DNA, totally or partially, from one end. In addition, binding of protein p6 to relaxed circular DNA induced positive supercoiling, indicating that a topological change in the DNA occurred.
Insights
Bacillus subtilis bacteriophage phi 29 protein p6 binds double-stranded DNA, particularly at terminal sequences. This interaction causes DNA protection and topological changes like positive supercoiling.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Bacteriophage phi 29 is a virus that infects Bacillus subtilis.
- Protein p6 is a key component of bacteriophage phi 29 involved in DNA packaging and replication.
- Understanding protein-DNA interactions is crucial for deciphering viral replication mechanisms.
Purpose of the Study:
- To investigate the DNA binding properties of bacteriophage phi 29 protein p6.
- To characterize the specific DNA sequences and structural changes associated with protein p6 binding.
- To elucidate the role of protein p6 in modulating DNA topology.
Main Methods:
- Gel retardation assays to assess DNA binding specificity.
- DNase I footprinting to map protein p6 binding sites on DNA.
- Electron microscopy to visualize protein p6-DNA complexes.
- Supercoiling assays to detect topological changes in DNA.
Main Results:
- Protein p6 exhibits specific binding to double-stranded DNA, with a preference for phi 29 terminal sequences.
- DNase I footprinting revealed characteristic protected regions and hypersensitive sites upon protein p6 binding.
- Electron microscopy confirmed that protein p6 covers DNA ends.
- Binding of protein p6 to relaxed circular DNA induced positive supercoiling.
Conclusions:
- Bacteriophage phi 29 protein p6 binds specifically to double-stranded DNA, particularly terminal sequences.
- Protein p6 binding alters DNA structure, leading to protection and topological changes such as positive supercoiling.
- These findings provide insights into the mechanism of DNA packaging and regulation by phi 29 protein p6.