Isomerization and apparent DNA bending by π, the replication protein of plasmid R6K

Krüger, Ricardo Henrique; Rakowski, Sheryl A.; Filutowicz, Marcin

doi:10.1016/j.bbrc.2003.12.022

Cited by 9 publications

(13 citation statements)

References 43 publications

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“…This may be a consequence of the known ability of protein to bend DNA (4,11,17,25). It has been shown that monomers and dimers bend a single iteron to similar degrees (ϳ50 o ) (17). Notably, when we conducted ligation enhancement assays with nonlimiting DNA concentrations, we were unable to see any significant differences in the amount of ligation between wt and copy-up .…”

Section: Vol 187 2005mentioning

confidence: 77%

“…4C). The reduced number of DNA-protein complexes (four) seen with the dimeric variant (His-⅐M36A ∧ M38A) could be explained based on previous EMSA experiments using His-⅐ M36A ∧ M38A and a two-DR-containing DNA fragment (17).…”

Section: Resultsmentioning

confidence: 99%

“…Additionally, we observed that compared to reactions containing ligase only, reactions containing ligase plus (wt or copy-up) show an approximately threefold increase in monomeric circles. This may be a consequence of the known ability of protein to bend DNA (4,11,17,25). It has been shown that monomers and dimers bend a single iteron to similar degrees (ϳ50 o ) (17).…”

Section: Vol 187 2005mentioning

confidence: 99%

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Role of π Dimers in Coupling (“Handcuffing”) of Plasmid R6K's γ ori Iterons

et al. 2005

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One proposed mechanism of replication inhibition in iteron-containing plasmids (ICPs) is "handcuffing," in which the coupling of origins via iteron-bound replication initiator (Rep) protein turns off origin function. In minimal R6K replicons, copy number control requires the interaction of plasmid-encoded protein with the seven 22-bp iterons of the ␥ origin of replication. Like other related Rep proteins, exists as both monomers and dimers. However, the ability of dimers to bind iterons distinguishes R6K from most other ICPs, where only monomers have been observed to bind iterons. Here, we describe experiments to determine if monomers or dimers of protein are involved in the formation of handcuffed complexes. Standard ligation enhancement assays were done using variants with different propensities to bind iterons as monomers or dimers. Consistent with observations from several ICPs, a hyperreplicative variant (⅐P106L ∧ F107S) exhibits deficiencies in handcuffing. Additionally, a novel dimer-biased variant of protein (⅐M36A ∧ M38A), which lacks initiator function, handcuffs iteron-containing DNA more efficiently than does wild-type . The data suggest that dimers mediate handcuffing, supporting our previously proposed model of handcuffing in the ␥ ori system. Thus, dimers of appear to possess three distinct inhibitory functions with respect to R6K replication: transcriptional autorepression of expression, in cis competition (for origin binding) with monomeric activator , and handcuffing-mediated inhibition of replication in trans.

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Section: Vol 187 2005mentioning

confidence: 77%

Section: Resultsmentioning

confidence: 99%

Section: Vol 187 2005mentioning

confidence: 99%

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Role of π Dimers in Coupling (“Handcuffing”) of Plasmid R6K's γ ori Iterons

et al. 2005

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“…There are several possible combinations of nucleoprotein complexes that can occur, and even a probe as simple as two direct repeats results in up to five shifted bands in the presence of WT protein (21). Thus, footprints in solution studies were expected to be composites of various families of nucleoprotein complexes, and the resulting data would likely be too complex to meaningfully address structure/function models of protein activities (in replication and transcription).…”

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confidence: 99%

Binding Modes of the Initiator and Inhibitor Forms of the Replication Protein π to the γ ori Iteron of Plasmid R6K

Kunnimalaiyaan

Krüger

Ross

et al. 2004

Journal of Biological Chemistry

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Discerning the interactions between initiator protein and the origin of replication should provide insights into the mechanism of DNA replication initiation. In the ␥ origin of plasmid R6K, the Rep protein, , is distinctive in that it can bind the seven 22-bp iterons in two forms; monomers activate replication, whereas dimers act as inhibitors. In this work, we used wild type and variants of the protein with altered monomer/dimer ratios to study iteron/ interactions. High resolution contact mapping was conducted using multiple techniques (missing base contact probing, methylation protection, base modification, and hydroxyl radical footprinting), and the electrophoretic separation of nucleoprotein complexes allowed us to discriminate between contact patterns produced by monomers and dimers. We also isolated iteron mutants that affected the binding of monomers (only) or both monomers and dimers. The mutational studies and footprinting analyses revealed that, when binding DNA, monomers interact with nucleotides spanning the entire length of the iteron. In contrast, dimers interact with only the left half of the iteron; however, the retained interactions are strikingly similar to those seen with monomers. These results support a model in which Rep protein dimerization disturbs one of two DNA binding domains important for monomer/iteron interaction; the dimer/iteron interaction utilizes only one DNA binding domain.

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“…The fraction of total DNA that was free, bound to one dimer, and bound to two dimers was quantified and plotted as a function of π concentration (Figure 3(d-f)). To calculate the cooperativity coefficient, k 12 , data in Figure 3 (d-f) were subjected to a least-squares linear regression analysis using equation (1c). This analysis provided the macroscopic binding constants, K 1 and K 2 , which were used to calculate k 12 using equation (2), as described previously.…”

Section: Quantification Of Cooperative π Dimer Binding To Adjacent Anmentioning

confidence: 99%

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

Bowers

Filutowicz

2008

Journal of Molecular Biology

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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...

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Isomerization and apparent DNA bending by π, the replication protein of plasmid R6K

Cited by 9 publications

References 43 publications

Role of π Dimers in Coupling (“Handcuffing”) of Plasmid R6K's γ ori Iterons

Role of π Dimers in Coupling (“Handcuffing”) of Plasmid R6K's γ ori Iterons

Binding Modes of the Initiator and Inhibitor Forms of the Replication Protein π to the γ ori Iteron of Plasmid R6K

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

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