HIGHLY COOPERATIVE DNA-BINDING BY THE COLIPHAGE HK022 REPRESSOR

The CI repressor protein from the temperate lambdoid phage HK022 was purified to near homogeneity and used in DNase I footprinting analyses to identify six binding sites in this phage. All these sites contained homologous 15 bp inverted repeats. Three of these 15 bp inverted repeats were located between the cI and cro (O(R)1 to O(R)3), and the other three were 3' to the cI gene (O(L)1 to O(L)3). Two of these sites were identified as operator sites for the repressor by DNA sequence analyses of virulent phage mutants. Almost all these mutations identified lay within the 15 bp inverted repeats comprising O(R)1 and O(R)2, and almost all were in the most highly conserved positions in the operators. The majority of virulent mutants contained mutations in both O(R)1 and O(R)2. Intrinsic affinities for individual operators were measured by DNase I footprinting analyses using DNA fragments which contained a single wild-type operator adjacent to two mutant operators. Comparison of these values with the affinity observed with these sites in the wild-type operator indicated that HK022 CI repressor bound cooperatively to O(R)1 and O(R)2 with a cooperativity parameter, Ω, of almost 2000. Cooperative binding occurred in an alternative pairwise fashion, as previously seen with λ CI repressor. In addition to cooperative binding between two adjacent operators, the repressor also increased the affinity for adjacent non-specific DNA sites, resulting in a periodic pattern of binding termed “phasing”. This phasing pattern extended beyond regions predicted for pair-wise interaction, but was significantly decreased on a template with two adjacent operators, suggesting that pairwise cooperativity interfered with phasing. © Academic Press Limited.