Highly cooperative DNA binding by the coliphage HK022 repressor

Area of Science:

• Molecular Biology
• Microbial Genetics
• Biochemistry

Background:

• The CI repressor protein is crucial for regulating gene expression in temperate lambdoid phages.
• Understanding phage-host interactions requires detailed knowledge of repressor-operator binding dynamics.

Purpose of the Study:

• To identify and characterize the DNA binding sites of the CI repressor from phage HK022.
• To investigate the cooperative binding mechanisms and DNA interaction patterns of the HK022 CI repressor.

Main Methods:

• Purification of the CI repressor protein from phage HK022.
• DNase I footprinting analyses to map repressor binding sites.
• DNA sequence analysis of virulent phage mutants to identify critical operator regions.
• Measurement of intrinsic operator affinities using modified DNA fragments.

Main Results:

• Six homologous 15 bp inverted repeat binding sites were identified for the HK022 CI repressor.
• Two operator sites (OR1 and OR2) were confirmed through mutant analysis, with mutations clustering in conserved regions.
• HK022 CI repressor exhibited strong cooperative binding to adjacent operators (OR1 and OR2) with a cooperativity parameter (omega) of approximately 2000.
• The repressor demonstrated cooperative binding in a pairwise fashion and induced a "phasing" effect on adjacent non-specific DNA sites.

Conclusions:

• The HK022 CI repressor displays complex cooperative binding behavior, similar to lambda CI repressor.
• Cooperative binding and phasing contribute to the precise regulation of phage gene expression.
• The interplay between pairwise cooperativity and phasing influences the overall DNA binding pattern.