Information analysis of sequences that bind the replication initiator RepA.

The replication initiator protein RepA of plasmid P1 can bind to 14 sites on the plasmid. These sites are variously used to autoregulate RepA synthesis and for initiation and control of DNA replication. Analysis of information (degree of conservation) at the sites revealed three sequence patches of high conservation. By saturation mutagenesis, the conservation at the outer two patches was found to contribute to RepA binding more critically. The guanine bases that are likely to contact RepA through the major groove were identified by methylation interference and methylation protection experiments. These bases mapped to the outer two patches and were separated by one turn of the helix. Therefore, they belong to major grooves on the same face of DNA. All backbone contacts of the protein, determined by hydroxyl radical footprinting, also mapped to the same face. We conclude from this that RepA binds to its site on one face of the DNA. Information analysis of binding sites for several prokaryotic repressors and activators, where the nature of DNA-protein contacts are known, revealed a correlation between the positions of high conservation and the positions of major grooves that faced the protein. The middle patch of high conservation in the RepA binding sites is an exception since in this region a minor groove is likely to face the protein. The simplest model for minor groove contacts suggests that in B-form DNA a T.A base-pair cannot easily be distinguished from an A.T pair by inspection of the minor groove. Yet in the RepA site, a T-->A mutation in the middle patch significantly affects binding. Therefore, the simplest models for both minor and major groove contacts are unlikely. It is possible that the patch determines the proper conformation of the site and thereby contributes to recognition indirectly.