Published online 9 June 2004
Nucleic Acids Research, 2004, Vol. 32, No. 10 3136-3147
© 2004 Oxford University Press
Recognition of DNA by 
protein from the broad-host range Streptococcus pyogenes plasmid pSM19035: analysis of binding to operator DNA with one to four heptad repeats
Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, 28049 Madrid, Spain, 1 Max-Delbrück-Centrum für Molekulare Medizin, 13092 Berlin, Germany, 2 Max-Planck-Institut für Molekulare Genetik, 14195 Berlin, Germany and 3 Institut für Kristallographie, Freie Universität Berlin, 14195 Berlin, Germany
*To whom correspondence should be addressed. Tel: +34 585 4546; Fax: +34 585 4506; Email: jcalonso{at}cnb.uam.es
Received February 19, 2004; Revised April 28, 2004;; Accepted May 13, 2004
pSM19035-encoded 
protein forms a dimer (2) that binds to a set of 7-bp repeats with sequence 5'-NATCACN-3'. Upon binding to its cognate sites, 2 regulates transcription of genes required for copy number control and stable inheritance of plasmids, and promotes accurate plasmid segregation. Protein 2 binds poorly to one heptad but the affinity to DNA increases with two and more unspaced heptads in direct or inverted orientation. DNA titration of increasing numbers of heptads with 2, monitored by circular dichroism measurements, indicates the binding of one 2 to one heptad (2:heptad stoichiometry of 1:1). Spacing of two directly or inversely oriented heptads by 1 to 7 bp reduces the affinity of the protein for its cognate target site. The binding affinity of 2 for two directly repeated heptads was severely reduced if one of the base pairs of the core 5'-ATCAC-3' sequence of one of the heptads was individually substituted by any other base pair. Hydroxyl radical footprinting shows a protection pattern at the 5'-ATCAC-3' core. These data suggest that each heptad defines an operator half-site and that tight binding of the symmetric 2 to the central 5'-TCA-3' core of symmetric or asymmetric targets (differently oriented heptads) is probably achieved by structural changes of DNA and/or protein or both.
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