Studies on the Binding of Lambda Int Protein to Attachment Site DNA: Identification of a Tight-binding Site in the P' Region

We have used three approaches to studying the interaction of lambda Int protein with bacteriophage attachment site DNA, POP': location of binding sites by retention of DNA fragments in a filter binding assay, reconstruction of a binding site by DNA synthesis and protection of a binding site from an exonuclease. Retention of restriction fragments on nitrocellulose filters in the presence of Int protein was used to locate binding sites. A high affinity binding site lies in P' between base pairs -6 and +173 from the center of the common core sequence, and low affinity sites are found in the 200 base pair region left of position -6. Reconstruction of the high affinity binding site region from the right using primed DNA synthesis and testing for filter binding in the presence of Int protein shows that sequences sufficient for tight binding of Int protein lie to the right of position +66. When attachment site DNA is protected by bound Int protein against digestion by exonuclease III, four Int dependent protection bands are seen in positions +58, +68, +79 and +88. This can be interpreted either as showing that four Int protein monomers bind to the high affinity region in series, or as evidence for wrapping of the DNA around Int protein, leading to structural changes resembling those occurring to DNA in nucleosomes.