Sequence-specific interactions of Rep proteins with ssDNA in the AT-rich region of the plasmid replication origin.
The DNA unwinding element (DUE) is a sequence rich in adenine and thymine residues present within the origin region of both prokaryotic and eukaryotic replicons. Recently, it has been shown that this is the site where bacterial DnaA proteins, the chromosomal replication initiators, form a specific nucleoprotein filament. DnaA proteins contain a DNA binding domain (DBD) and belong to the family of origin binding proteins (OBPs). To date there has been no data on whether OBPs structurally different from DnaA can form nucleoprotein complexes within the DUE. In this work we demonstrate that plasmid Rep proteins, composed of two Winged Helix domains, distinct from the DBD, specifically bind to one of the strands of ssDNA within the DUE. We observed nucleoprotein complexes formed by these Rep proteins, involving both dsDNA containing the Rep-binding sites (iterons) and the strand-specific ssDNA of the DUE. Formation of these complexes required the presence of all repeated sequence elements located within the DUE. Any changes in these repeated sequences resulted in the disturbance in Rep-ssDNA DUE complex formation and the lack of origin replication activity in vivo or in vitro.
Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology, University of Gdansk, 24 Kladki, 80-822 Gdansk, Poland.
Katarzyna
K
Wegrzyn
Department of Macromolecular Structures, Centro Nacional de Biotecnologia, CSIC, Darwin 3, 28049 Cantoblanco, Madrid, Spain.
Maria Eugenia
ME
Fuentes-Perez
Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology, University of Gdansk, 24 Kladki, 80-822 Gdansk, Poland.
Katarzyna
K
Bury
Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology, University of Gdansk, 24 Kladki, 80-822 Gdansk, Poland.
Magdalena
M
Rajewska
Department of Macromolecular Structures, Centro Nacional de Biotecnologia, CSIC, Darwin 3, 28049 Cantoblanco, Madrid, Spain igor.konieczny@biotech.ug.edu.pl.
Fernando
F
Moreno-Herrero
Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology, University of Gdansk, 24 Kladki, 80-822 Gdansk, Poland igor.konieczny@biotech.ug.edu.pl.
Igor
I
Konieczny
D020856:AT Rich Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D018741:Replication Origin
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins