Structural Changes in RepA, a Plasmid Replication Initiator, upon Binding to Origin DNA

Abstract

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.

Transition from a plasmid to a chromosomal mode of replication entails additional regulators

“…The 39-mer most likely acts as a DNA chaperone that remodels RctB to a form that enhances protein-protein interactions involved in handcuffing. Protein remodeling upon interaction with DNA is well known (22)(23)(24). It has been shown also for the two initiators of the iteron-bearing plasmids (23,25) and for the E. coli initiator (26).…”

“…Protein remodeling upon interaction with DNA is well known (22)(23)(24). It has been shown also for the two initiators of the iteron-bearing plasmids (23,25) and for the E. coli initiator (26). Increased handcuffing could explain the potency of 39-mer-carrying fragments in reducing oriII copy number (Fig.…”

New pSC101-derivative cloning vectors with elevated copy numbers

“…The side chain of E93 is circled and is located in β2b, a β-sheet important for protein dimerization. The structure of RepE (Diaz-Lopez et al, 2003) was obtained from PDB file 1REP and imaged with the program Cn3D. Helical regions are represented as cylinders and β-sheets as ribbons.…”

RepA-WH1prionoid

“…15 Allosteric binding of the origin of replication-specific dsDNA sequence to the RepA dimers exerts a conformational change that, besides dissociating the protein in its constituent monomers, refolds two α-helical stretches in RepA-WH1 as β-strands and loops, enabling both WH1 and WH2 to bind to the iteron sequence. [16][17][18] By binding to the iteron repeats, RepA monomers build a nucleoprotein complex that initiates plasmid replication, de-stabilizing the DNA double helix and attracting to the origin the required host replication factors (helicase, primase, DNA polymerase). Once a full replication round is completed, the WH1 domain in RepA monomers is involved in negative regulation of DNA replication, to keep plasmid copy-number within control: interactions between WH1 domains in RepA monomers bound to two distinct, recently replicated plasmid molecules build the axial core of a nucleoprotein filament which curb new rounds of premature origin firing.…”

“…21 However, it was located at the C-terminus of an α-helix (α2) in the crystal structure of dimeric RepA-WH1, 17 partially buried in the hydrophobic core of the domain but becoming exposed to the solvent in the monomeric species. 14,16 The score for the aggregation prediction microorganisms have been instrumental in addressing the molecular basis for amyloid conformational templating, structural polymorphism and cell-to-cell transmissibility. [3][4][5] However, two limitations to the applicability of yeast prions as universal models for amyloidosis are noteworthy: (1) the amyloidogenic sequence stretches in yeast prions are consistently Gln/Asn-rich, unlike most proteins involved in amyloid proteinopathies (which bear hydrophobic stretches) with the exception of the proteins involved in Huntington disease and in related ataxias; (2) even more importantly, while yeast prions are the epigenetic determinants of distinct, mildly advantageous phenotypes that improve adaptability to environmental challenges, 6,7 they are not the causative agents of a proteinopathy in yeast albeit, when overexpressed, Sup35p/[PSI + ] becomes detrimental for cell growth.…”