The bacteriophage phi29 packaging proteins supercoil the DNA ends

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Bacteriophage phi29 DNA replication involves a terminal protein (DNA-gp3) covalently attached to the DNA ends.
• DNA packaging into bacteriophage heads is a complex process requiring specific viral proteins.

Purpose of the Study:

• To investigate the role of terminal protein (DNA-gp3) and packaging ATPase (gp16) in condensing and organizing bacteriophage phi29 DNA.
• To elucidate the structural changes in DNA-gp3 upon interaction with gp16 and their implications for DNA packaging.

Main Methods:

• Sucrose density gradient sedimentation to assess DNA-protein complex formation and compactness.
• Electron microscopy to visualize the structure of DNA-protein complexes.
• In vitro packaging assay using a defined system with proheads.
• Topoisomerase I treatment to probe DNA supercoiling.

Main Results:

• DNA-gp3 and its fragments sedimented faster than proteinase K-treated counterparts, indicating increased compactness dependent on gp3 and Mg2+.
• Addition of gp16 further increased sedimentation rates and condensed DNA-gp3, with [35S]gp16 cosedimenting with DNA.
• Electron microscopy revealed lariat structures for DNA-gp3, which became more complex with multiple loops in the presence of gp16, consistent with supercoiling.

Conclusions:

• Bacteriophage phi29 DNA-gp3 undergoes a maturation pathway involving supercoiling of DNA ends by gp3 and gp16.
• This DNA supercoiling likely facilitates efficient interaction with the bacteriophage prohead during packaging.
• The combined action of gp3 and gp16 leads to DNA condensation and organization essential for viral DNA packaging.