Early Events in the Binding of the pPS10 Replication Protein RepA to Single Iteron and Operator DNA Sequences

Díaz-López, Teresa; Dávila-Fajardo, Cristina Lucía; Blaesing, Franca; Lillo, M. Pilar; Giraldo, Rafael

doi:10.1016/j.jmb.2006.09.013

Cited by 32 publications

(46 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The occurrence of just a few intermolecular contacts, in a quasi-native folding intermediate, would be sufficient to drive the assembly of amyloids (25)(26)(27)(28). In the light of the previous work on DNA-induced transformation of RepA (20,22,23), there is an inverse correlation between the magnitude of the structural change elicited by each dsDNA sequence (iteron Ն nonspecific Ͼ operator) and the internal order of the resulting WH1-A31V assemblies (irregular Ͻ spheroids Ͻ fibrils). The exposure to the solvent of hydrophobic residues in WH1, associated with 5Јiter binding and monomerization (19), must lead to uncontrolled aggregation.…”

Section: Discussionmentioning

confidence: 97%

“…WH1 amyloidogenic stretch is part of a very stable subdomain (␣1-␣2-␣5) that brings together both protein ends (20). Thus, a ''three-dimensional domain-swapped'' type of assembly (13) seems unlikely, because it would imply extensive unfolding/refolding, the kind of structural transformation that occurs upon iteron-induced monomerization, not upon operator binding (19)(20)(21)(22)(23).…”

Section: Discussionmentioning

confidence: 99%

“…Because the binding of each of the individual RepA-WH domains to their target sequences has very low affinity and is expected to be transient (21,23), the fibrillar assemblies were analyzed to establish whether they contained dsDNA. First, ultracentrifugation of mature fiber preparations containing equimolar concentrations of opsp dsDNA and WH1-A31V yielded DNA essentially in the soluble fraction (Fig.…”

Section: Dna Is Not a Component Of Wh1-a31v Fibrillar Assembliesmentioning

confidence: 99%

See 2 more Smart Citations

Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures

Giraldo

2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

171

View full text Add to dashboard Cite

RepA, the replication initiator protein of Pseudomonas pPS10 plasmid, is made of two winged-helix (WH) domains. RepA dimers undergo a structural transformation upon binding to origin DNA sequences (iterons), resulting in monomerization and ␣-helix into ␤-strand conversion. This affects the N-terminal domain (WH1) and generates a metastable intermediate. Here it is shown that the interaction of short dsDNA oligonucleotides, including iteron or operator RepA targets, with the isolated WH1 domain promotes the assembly of different nanostructures. These range from irregular aggregates to amyloid spheroids and fibers. Their intrinsic order inversely correlates with the extent of the transformation induced by each DNA sequence on RepA. However, DNA is not a constituent of the assembled fibers, in agreement with the protein-only principle for amyloid structure. Thus, the RepA-WH1 domain on DNA binding mimics the behavior of the mammalian prion protein. The stretch of amino acids responsible for WH1 aggregation has been identified, leading to the design of mutants with enhanced or reduced amyloidogenicity and the synthesis of a peptide that assembles into a cross-␤ structure. RepA amyloid assemblies could have a role in the negative regulation of plasmid replication. This article underlines the potential of specific nucleic acid sequences in promoting protein amyloidogenesis at nearly physiological conditions. amyloidogenesis ͉ conformational changes ͉ DNA binding ͉ fiber assembly ͉ RepA protein T he formation of protein assemblies with amyloid properties is a subject of intense research because of their involvement in severe human diseases (1, 2). An unsolved issue is the nature of the signal(s) responsible for amyloidogenesis, which includes proteolytic processing of a functional precursor and mutations, and also hyperphosphorylation (3) or the binding to elements of the cell envelope (4) or nucleic acids (5-8). The major (if not the only) component of any mature amyloid assembly is protein (9, 10). The studies of amyloidogenesis have been extended to model systems based in proteins not involved in any disease (1).Our current understanding of protein aggregation into amyloids suggests that some partially unfolded intermediates that populate the funnel-shaped folding energy landscape of virtually any protein aggregate into a stable, low-energy conformation distinct from the protein native state (1, 2). Supramolecular assembly is a common functional resource for proteins by using an ample repertoire of protein-protein interfaces. However, the family of structures responsible for the unique staining (11) and diffraction (12) properties of any protein amyloid is known as cross-␤: a ␤-sheet of indefinite length is formed by the stacking, orthogonal to its axis, of ␤-strands from each protein monomer (13). Amyloidogenesis often includes the refolding as ␤-strands of protein regions that, although compatible with such secondary structure, are found as ␣-helices or coils in the native structure (14-18). In other cases, nearly...

show abstract

Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Section: Dna Is Not a Component Of Wh1-a31v Fibrillar Assembliesmentioning

confidence: 99%

See 1 more Smart Citation

Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures

Giraldo

2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

171

View full text Add to dashboard Cite

show abstract

“…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.…”

Section: Repa-wh1 Prionoidmentioning

confidence: 99%

RepA-WH1prionoid

Giraldo

Espina

Fernández-Tresguerres

et al. 2011

Prion

Self Cite

View full text Add to dashboard Cite

“…9 Since then, two more Reps were also shown or inferred to bind iterons as dimers, thus establishing a new trend in Rep/iteron control. [21][22][23] Although a π dimer can bend iteron DNA to the same extent as a π monomer, 12 π dimers cannot induce DNA strand separation, 16 yet the binding of π dimers to iterons has several consequences for γ ori replication control. 9,15-17 A π monomer contacts both the 5' half and the 3' half of the iteron through the C-terminal winged-helix (WH2) and the N-terminal winged helix (WH1), respectively, but a π dimer only contacts the 5' half of each iteron, including a highly conserved TGAGnG motif, with the WH2 of one of its subunits.…”

mentioning

confidence: 99%

Cooperative Binding Mode of the Inhibitors of R6K Replication, π Dimers

Bowers

Filutowicz

2008

Journal of Molecular Biology

View full text Add to dashboard Cite

The replication initiator protein, π, plays an essential role in the initiation of replication of plasmid R6K. Both monomers and dimers of π bind to iterons in the γ origin of plasmid R6K, yet monomers facilitate open complex formation while dimers, the predominant form in the cell, do not. Consequently, π monomers activate replication while π dimers inhibit replication. Recently, it was shown that the monomeric form of π binds multiple tandem iterons in a strongly cooperative fashion, which might explain how monomers out-compete dimers for replication initiation when plasmid copy number and π supply are low. Here, we examine cooperative binding of π dimers and explore the role these interactions may have in the inactivation of γ origin. To examine π dimer/iteron interactions in the absence of competing π monomer/iteron interactions using wild-type π, constructs were made with key base changes to each iteron that eliminate π monomer binding yet have no impact on π dimer binding. Our results indicate that in the absence of π monomers, π dimers bind with greater cooperativity to alternate iterons than adjacent iterons, thus preferentially leaving intervening iterons unbound and the origin unsaturated. We discuss new insights into plasmid replication control by π dimers.It is believed that all naturally occurring plasmids employ efficient copy-control mechanisms to ensure their maintenance at a reasonably constant copy number from cell to cell. The antibiotic resistance plasmid, R6K, is maintained at a steady 15-20 copies per chromosome 1 in a wide variety of bacterial hosts. 2 For this to occur, regulatory controls at the step of replication initiation work to increase low plasmid copy numbers and reduce elevated ones. 3Controlled replication of plasmid R6K requires two plasmid-encoded elements: the iterons in the γ origin of replication (γ ori) and the pir gene that encodes the replication (Rep) protein, π ( Figure 1) 4-8 γ ori activation requires the binding of monomers of π protein to the seven 22 base pair (bp) iterons within γ ori that are adjacent to an A+T-rich region of the plasmid. 9-11 The binding of π monomers to iterons causes an apparent bending of the origin DNA, allowing the nearby A+T-rich region to melt, the replication complex to bind, and replication to start uni-directionally from a specific site within the A+T-rich region. 11-13While monomers of π activate replication, dimers of π appear to inhibit replication through several different mechanisms (Figure 1). 9,14-17 π dimers bind a non-iteron site within the A +T-rich region in proximity to the start sites for leading strand synthesis. 18 It has been hypothesized that π dimers negatively modulate the priming step of the replication process by *Corresponding author (M. Filutowicz):Tel. 608-262-6947; Fax. 608-262-9865; E-mail: msfiluto@facstaff.wisc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manu...

show abstract

Early Events in the Binding of the pPS10 Replication Protein RepA to Single Iteron and Operator DNA Sequences

Cited by 32 publications

References 41 publications

Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures

Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures

RepA-WH1prionoid

Cooperative Binding Mode of the Inhibitors of R6K Replication, π Dimers

Contact Info

Product

Resources

About