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J. Biol. Chem., Vol. 278, Issue 20, 18606-18616, May 16, 2003
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From the Departments of RepA protein is the DNA
replication initiator of the Pseudomonas plasmid pPS10.
RepA dimers bind to an inversely repeated operator sequence in
repA promoter, thus repressing its own synthesis, whereas
monomers bind to four directly repeated sequences (iterons) to initiate
DNA replication. We had proposed previously that RepA is composed of
two winged-helix (WH) domains, a structural unit also present in
eukaryotic and archaeal initiators. To bind to the whole iteron
sequence through both domains, RepA should couple monomerization
to a conformational change in the N-terminal WH, which includes a
leucine zipper-like sequence motif. We show for the first time
that, by itself, binding to iteron DNA in vitro dissociates
RepA dimers into monomers and alters RepA conformation, suggesting an
allosteric effect. Furthermore, we also show that similar changes in
RepA are promoted by mutations that substitute two Leu residues of the
putative leucine zipper by Ala, destabilizing the hydrophobic core of
the first WH. We propose that this mutant (RepA-2L2A) resembles a
transient folding intermediate in the pathway leading to active
monomers. These findings, together with the known activation of other
Rep-type proteins by chaperones, are relevant to understand the
molecular basis of plasmid DNA replication initiation.
Structural Changes in RepA, a Plasmid Replication
Initiator, upon Binding to Origin DNA*
,§,,, and
Molecular Microbiology and
§ Protein Structure and Function and ¶ Analytical
Ultracentrifugation Facility, Centro de Investigaciones
Biológicas, Consejo Superior de Investigaciones
Científicas (CSIC), C/Velázquez, 144, 28006 Madrid,
Spain
*
This work was supported by grants from the Spanish
Comisión Interministerial de Ciencia y Tecnología (PM99-0096)
and Red Temática de Investigación Cooperativa (FIS C03/14).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
34-91-5611800; Fax: 34-91-5627518; E-mail: rgiraldo@cib.csic.es.
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