Characterization of the interaction between the lambda intasome and attB

Area of Science:

• Molecular Biology
• Genetics
• Virology

Background:

• Bacteriophage lambda DNA integrates into Escherichia coli chromosomes via an intasome complex.
• This complex, comprising integrase (Int) and integration host factor (IHF), locates and binds bacterial attachment sites (attB) to facilitate integration.

Purpose of the Study:

• To characterize the interaction between the bacteriophage lambda intasome and the attB site.
• To determine the dissociation constant (Kd) of the intasome-attB complex.
• To investigate the role of attB core binding sites in intasome capture and integration efficiency.

Main Methods:

• Measuring integration reaction rates as a function of attB DNA concentration to determine saturation constants.
• Using a competitive inhibition assay with a modified attB site to determine inhibition constants.
• Comparing the binding affinity of wild-type Int/IHF with a mutant integrase (IntE174K).

Main Results:

• The dissociation constant for the intasome-attB complex was determined to be approximately 300 nM using two independent methods.
• Both core binding sites within attB are essential for efficient intasome capture.
• A mutant integrase (IntE174K) exhibited significantly lower dissociation constants, indicating increased affinity for core sites and potential IHF-independent function.

Conclusions:

• The intasome-attB interaction is characterized by a dissociation constant of ~300 nM, with both core sites being critical for binding.
• The increased affinity of IntE174K for attB core sites explains its ability to function without IHF.
• The sequence-specific nature of attB inhibition suggests complex regulatory mechanisms in phage DNA integration.