Defining cosQ, the site required for termination of bacteriophage lambda DNA packaging

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• Bacteriophage lambda utilizes specific DNA recognition sites (cos) for packaging concatemeric DNA into virion chromosomes.
• The cos site comprises three subsites: cosN (nicking), cosB (packaging initiation), and cosQ (packaging termination).
• cosQ is implicated in delivering terminase for nicking the bottom strand of cosN during packaging termination.

Purpose of the Study:

• To precisely define the functional segment of cosQ involved in bacteriophage lambda chromosome packaging termination.
• To investigate the mechanism by which cosQ facilitates terminase delivery and nicking at cosN.
• To elucidate the role of sequence and spacing within the cos site for efficient packaging.

Main Methods:

• Saturation mutagenesis was employed to identify the critical nucleotides within the cosQ site.
• Double cosQ mutants and substitutions within the inter-site region were constructed and analyzed.
• Insertional mutagenesis was used to assess the importance of spacing between cosQ and cosN.

Main Results:

• Saturation mutagenesis identified a 7-bp segment as the functional cosQ site.
• Mutational analysis refuted a model requiring sequence homology between cosQ and cosNR.
• Insertional mutagenesis demonstrated that precise spacing between cosQ and cosN is crucial for function.
• Lethal helical insertions disproved a DNA looping model for terminase delivery.

Conclusions:

• The 7-bp cosQ site is essential for bacteriophage lambda chromosome packaging termination.
• cosQ function is independent of sequence complementarity with cosNR, challenging previous models.
• Proper spatial arrangement between cosQ and cosN is critical, and DNA looping is not the mechanism for terminase delivery.
• The cosQ recognition sequence is identical to that of the EcoO109I restriction enzyme.