Binding and bending of the lambda replication origin by the phage O protein.

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Bacteriophage lambda DNA replication is initiated by the O protein.
• The phage origin of replication contains multiple iteron sequences.

Purpose of the Study:

• To characterize the binding of lambda phage replication initiation protein O to its origin of replication.
• To determine the minimal DNA sequence required for O protein binding.
• To investigate the structural consequences of O protein-DNA interaction.

Main Methods:

• Electrophoretic mobility shift assays (EMSAs)
• Ethylation interference assays
• Dimethyl sulfate protection assays
• Neocarzinostatin footprinting
• DNA bending analysis
• Cylindrical DNA projection modeling

Main Results:

• The minimal DNA binding site for O protein is a single iteron, a 19-bp sequence with dyad symmetry.
• The N-terminal domain of O protein is sufficient for DNA binding and induces DNA bending.
• DNA-protein contacts are concentrated symmetrically, suggesting dimer binding, but some asymmetric contacts indicate helix distortion.
• Modeling reveals O protein binding differs from lambda repressor binding, leaving the major groove exposed.

Conclusions:

• Lambda phage replication protein O binds to a specific iteron sequence within the origin of replication.
• O protein binding induces DNA bending and helix distortion, potentially facilitating replication initiation.
• The binding mode of O protein is distinct from that of lambda repressor.