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J. Biol. Chem., Vol. 276, Issue 13, 10234-10246, March 30, 2001

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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 pAM1 from Gram-positive Bacteria^*

Emmanuelle Le Chatelier, Laurent Jannière, S. Dusko Ehrlich, and Danielle Canceill^§

From the Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78350 Jouy en Josas, France

The RepE protein of the broad host range pAM1 plasmid from Gram-positive bacteria is absolutely required for replication. To elucidate its role, we purified the protein to near homogeneity and analyzed its interactions with different nucleic acids using gel retardation assays and footprinting experiments. We show that RepE is monomeric in solution and binds specifically, 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°). Unexpectedly, RepE also binds nonspecifically to single-stranded DNA with a 2-4-fold greater affinity than for double-stranded origin. On a supercoiled plasmid, RepE binding to the 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 supercoiling are required), and (iii) its melted region serves as a substrate for RepE binding. These original properties together with the fact that pAM1 replication depends on a transcription step through the origin on DNA polymerase I to initiate replication and on a primosome to load the replisome suggest that the main function of RepE is to assist primer generation at the origin.

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