The RepE initiator is a double-stranded and single-stranded DNA-binding protein that forms an atypical open complex at the onset of replication of plasmid pAM beta 1 from gram-positive bacteria

The RepE protein of the broad host range pAM beta1 plasmid from Gram-positi ve bacteria is absolutely required for replication. To elucidate its role, we purified the protein to near homogeneity and analyzed its interactions w ith different nucleic acids using gel retardation assays and footprinting e xperiments. We show that RepE is monomeric in solution and binds specifical ly, rapidly, and durably to the origin at a unique double-stranded binding site immediately upstream from the initiation site of DNA replication. The binding induces only a weak bend (31 degrees), Unexpectedly, RepE also bind s nonspecifically to single-stranded DNA with a 2-4-fold greater affinity t han for double-stranded origin. On a supercoiled plasmid, RepE binding to t he double-stranded origin leads to the denaturation of the AT-rich sequence immediately downstream from the binding site to form an open complex. This open complex is atypical since (i) its formation requires neither multiple RepE binding sites on the double-stranded origin nor strong bending of the origin, (ii) it occurs in the absence of any cofactors (only RepE and supe rcoiling are required), and (iii) its melted region serves as a substrate f or RepE binding. These original properties together with the fact that pAM beta1 replication depends on a transcription step through the origin on DNA polymerase I to initiate replication and on a primosome to load the replis ome suggest that the main function of RepE is to assist primer generation a t the origin.