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The interaction of DNA with bacteriophage phi 29 connector: a study by AFM and TEM
M Valle1, J M Valpuesta, J L Carrascosa
1Centro Nacional de Biotecnología, CSIC, Universidad Autónoma de Madrid, Cantoblanco, Spain.
Abstract:
The connector of bacteriophage phi 29 is involved in DNA packaging during viral morphogenesis and we have studied its in vitro binding to DNA using either linear or circular DNA. The protein-DNA complexes have been analyzed by transmission electron microscopy (TEM) and by atomic force microscopy (AFM) of samples directly deposited on mica. TEM showed the presence of a specific binding due to the interaction of the protein with the free ends of the DNA. The study of these samples by AFM showed two major types of morphologies: The interaction of the connector with circular DNA revealed that the strands of DNA that enter and exit the protein complex form an angle with a mean value of 132 degrees. Nevertheless, when the connector was incubated with linear DNA (and later circularized), there was an additional bend angle of about 168 degrees. Further morphological analysis of the latter samples by AFM revealed a structure of the protein-DNA complex consistent with the DNA traversing the connector, probably through the inner channel. On the other hand, images from the samples obtained by incubation of the connector with circular DNa were consistent with an interaction of the DNA with the outer side of the connector.
Insights
The bacteriophage phi 29 connector protein binds DNA ends. Atomic force microscopy reveals DNA traverses the connector channel or interacts externally, influencing DNA packaging during viral assembly.
Area of Science:
- Molecular Biology
- Virology
- Biophysics
Background:
- The bacteriophage phi 29 connector protein is crucial for DNA packaging during viral replication.
- Understanding its interaction with DNA is key to viral morphogenesis.
Purpose of the Study:
- To investigate the in vitro binding of the bacteriophage phi 29 connector protein to both linear and circular DNA.
- To elucidate the structural basis of DNA-protein interactions using advanced microscopy techniques.
Main Methods:
- Transmission Electron Microscopy (TEM) for visualizing protein-DNA complexes.
- Atomic Force Microscopy (AFM) for high-resolution morphological analysis of deposited samples.
- In vitro incubation of connector protein with linear and circular DNA.
Main Results:
- TEM confirmed specific binding of the connector protein to the free ends of DNA.
- AFM revealed distinct morphologies: circular DNA interacted externally, while linear DNA (circularized) showed traversal through the connector channel.
- DNA bending angles were measured: 132 degrees for external interaction and 168 degrees for channel traversal.
Conclusions:
- The bacteriophage phi 29 connector protein exhibits differential binding modes with DNA depending on its form (linear vs. circular).
- DNA traversal through the connector's inner channel is a likely mechanism for packaging.
- These findings provide structural insights into viral DNA packaging mechanisms.