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DNA structure in the nucleoprotein complex that activates replication of phage phi 29
J M Hermoso1, R Freire, A Bravo
1Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain.
Biophysical Chemistry
|May 1, 1994
Summary
Viral protein p6 initiates phage phi 29 DNA replication by forming a nucleoprotein complex. This complex features a right-handed DNA superhelix around a protein core, with specific helical parameters and a DNA-binding domain identified.
Area of Science:
- Molecular Biology
- Virology
- Structural Biology
Background:
- Phage phi 29 DNA replication initiation relies on viral protein p6.
- Protein p6 forms a nucleoprotein complex at the genome's replication origins.
- This complex involves a DNA right-handed superhelix around a multimeric protein core.
Purpose of the Study:
- To elucidate the superhelical path of DNA within the p6 nucleoprotein complex.
- To identify the DNA binding domain of protein p6.
- To correlate in vitro findings with in vivo replication activation properties.
Main Methods:
- Determination of DNA superhelical path using linking number changes.
- Measurement of surface-related helical repeat and DNA compaction.
- Site-directed mutagenesis to analyze protein p6 mutants.
Main Results:
- One superhelical turn of DNA corresponds to approximately 63 base pairs (2.6 p6 dimers).
- The DNA binding domain of protein p6 is located in the N-terminal region, predicted to be an amphipathic alpha-helix.
- Mutants in the polar side of this helix showed impaired DNA binding and replication activation.
Conclusions:
- The study defines the DNA superhelical structure within the phage phi 29 replication initiation complex.
- The N-terminal amphipathic alpha-helix of protein p6 is crucial for DNA binding and replication activation.
- These findings provide insights into viral DNA replication mechanisms.