The bacteriophage lambda terminase. Partial purification and preliminary characterization of properties

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Bacteriophage lambda DNA maturation and packaging into infectious phage particles are controlled by the Nu1 and A genes.
• Efficient packaging requires specific protein factors encoded by these genes.

Purpose of the Study:

• To purify and characterize the protein responsible for bacteriophage lambda DNA packaging and cohesive-end site cleavage.
• To elucidate the enzymatic activities and subunit composition of this key protein.

Main Methods:

• Extensive protein purification using adsorption, ion exchange, and affinity chromatography.
• Enzyme activity assays for DNA packaging and ATP hydrolysis.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for purity assessment and molecular weight determination.

Main Results:

• A single protein, terminase, was purified 60,000-fold and shown to be 30-80% homogeneous.
• Terminase catalyzes both lambda DNA packaging and endonucleolytic cleavage of the cohesive-end site, both ATP-dependent reactions.
• Purified terminase exhibits DNA-dependent ATPase activity and has a native molecular weight of 117,000, likely composed of gpA (74,000 Da) and two gpNu1 (21,000 Da) subunits.

Conclusions:

• Terminase is the key enzyme complex responsible for bacteriophage lambda DNA packaging and processing.
• The enzyme's dual function and subunit composition provide insights into the mechanism of phage DNA maturation and packaging.