Effect of supercoiling on the λ switch

Article Addendum Article Addendum Bacteriophage 4, e27517; January–December 2014; © 2014 Landes Bioscience Effect of supercoiling on the λ switch Kamilla Norregaard,1 Magnus Andersson,1,† Kim Sneppen,1 Peter Eigil Nielsen,2 Stanley Brown,1 and Lene B Oddershede1,* The Niels Bohr Institute; University of Copenhagen; Copenhagen, Denmark; 2Department of Cellular and Molecular Medicine; Faculty of Health and Sciences; 1 Copenhagen, Denmark † Current affiliation: Department of Physics; Umeå University; Umeå, Sweden T he lysogenic state of the λ switch is exceptionally stable, still, it is capa- ble of responding to DNA-damage and causing the prophage to enter lytic devel- opment. In strains lacking RecA protein, three quarters of the released phage bear rapidly enter the lytic state. We invented mutations in the cI gene.2 Thus, the pro- an assay where PNA mediated tethering phage state maintained by CI is more sta- of a plasmid allowed for single molecule ble than the genes encoding components investigations of the effect of supercoil- maintaining the repressed state. In fact, ing on the efficiency of the epigenetic two more recent studies found 73 out of λ switch. Compared with non-super- a total of 74 apparently wild type phage coiled DNA, the presence of supercoils (cI+) released from recA lysogens, were enhances the CI-mediated DNA loop- mutants in the promoter for repressor ing probability and renders the transi- maintenance, pRM.3,4 The rarity of wild tion between the looped and unlooped type phage released from recA lysogens, as states steeper, thus increasing the Hill pointed out by Little and Michaelowski,4 coefficient. Interestingly, the transition may not even be true escapees but may occurs exactly at the CI concentration be the result of a mutation in the now corresponding to the minimum number dead host. The stability of the prophage of CI molecules capable of maintaining state is in part due to the sophisticated the pRM-repressed state. Based on these regulation of CI synthesis (as detailed in results we propose that supercoiling Fig. 1B). The intracellular concentration maintains the pRM-repressible state as of CI must be carefully fine-tuned such CI concentration decline during induc- that it is high enough to maintain repres- tion and thus prevent autoregulation of sion of lytic development but not too cI from interfering with induction. high to prevent sufficient degradation by Keywords: supercoiling, λ switch, activated RecA.5 In the lysogenic state, CI epigenetics, PNA, cooperativity, CI Introduction activates its own synthesis from pRM.6 protein, tethered particle motion At physiological concentrations CI can Submitted: 11/08/2013 The lysis–lysogeny decision by bac- repress its own synthesis by binding to Revised: 12/12/2013 teriophage λ was the first genetic switch the operator, OR3, only if another seg- Accepted: 12/12/2013 to be deciphered,1 this epigenetic switch ment of the prophage genome contain- is now relatively well understood and the ing the operator OL3 is clamped next to http://dx.doi.org/10.4161/bact.27517 most important features are outlined in OR3. The clamp is formed by CI binding *Correspondence to: Lene B Oddershede; Figure 1A and B. One fascinating fea- as an octamer to the operators OL1-OL2 Email: [email protected] ture of this switch is the profound stabil- and OR1-OR2, thus arranging the inter- ity of the prophage state. The λ prophage vening DNA in a loop (as depicted in Addendum to: Norregaard K, Andersson M, Sneppen K, Nielsen PE, Brown S, Oddershede LB. responds to the host (Escherichia coli) Fig. 1B).7 In other words, the OL-CI-OR DNA supercoiling enhances cooperativity and DNA-damage sensing or SOS system. looped state is necessary for CI repression efficiency of an epigenetic switch. Proc Natl Acad When the SOS pathway is induced, of pRM. Sci U S A 2013; 110:17386–91; PMID:24101469; activated RecA protein catalyzes the CI-mediated looping of DNA has http://dx.doi.org/10.1073/pnas.1215907110 self-cleavage of λ repressor protein, CI, been extensively studied in vivo and www.landesbioscience.com Bacteriophage e27517-1  Figure 1. (A) λ phage survival strategies after infection of Escherichia coli. The phage enters either the lytic pathway (left) or the lysogenic pathway (right). The lytic pathway is irreversible and rapidly produces a crop of phage that is released by lysing the cell. The lysogenic pathway is a dormant state in which the phage DNA is incorporated into the host DNA and passively gets replicated until a signal (e.g., DNA damage) flips the switch and causes entering into the lytic state. (B) λ switch regulation. The λ operators, operator right (OR) and operator left (OL), are located ~2.3 kbp apart on the phage DNA overlapping the lysogenic (pRM) and lytic (pR and pL) promoters (marked by bent arrows). Each operator is a constellation of three adjacent sub sites that bind CI (yellow dumbbell) in a hierarchical manner. Cooperative binding between CI dimers bound at OR1 and the adjacent intrinsically weak operator OR2 virtually ensures simultaneously occupancy and is responsible for lytic repression (indicated by red cross) and simultaneously activation of the weak CI promoter pRM located at OR3. Long-range cooperativity affords increased stability to the lysogenic state by a CI octamer clamping OR1-OR2 and OL1-OL2 arranging the intervening DNA in a loop. This complex brings OL3 and OR3 in juxtaposition allowing a CI dimer bound on the intrinsic strong OL3 to assist a CI dimer binding at the weak OR3 resulting in pRM repression (marked by red cross). (C) The PNA-based TPM assay. A DNA plasmid is tethered between an anti-digoxigenin coated glass surface and a streptavidin coated polystyrene bead via digoxigenin/biotin PNA handles that form triplex invasion complexes with specific sequences on the DNA. The non-complementary strand is displaced as a small loop. The PNA handles are flanking the λ operators, OR and OL, which limit the λ immunity region. in vitro. In vitro it was studied both in TPM studies were performed with lin- PNA Tethering bulk and single molecule experiments,8,9 ear DNA with no supercoils unless they of Supercoiled DNA where the most common single molecule were mechanically introduced, e.g., by approach employed was an assay where a twisting a magnetic bead. Mechanically Recently, we developed a novel linear DNA was tethered between a cov- induced supercoils can be difficult to chemical/biological assay for investigat- erslip and a submicron sized bead whose distinguish from protein-mediated ing protein-mediated DNA looping at Brownian motion was observed and ana- looping events. In this addendum, we a single molecule level using naturally lyzed. The larger the excursions of the describe a novel TPM assay utilizing supercoiled DNA.10 Our assay (shown in particle, the longer the tether and the peptide nucleic acid (PNA) handles Fig. 1C) consists of a single supercoiled looped state (yielding an overall shorter to tether natively supercoiled plasmid plasmid tethered by sequence specific DNA tether) could thereby be distin- DNA.10 We discuss, to our knowledge, PNA handles allowing us to tag points guished from the unlooped state. This the first single molecule evidence of the on circular DNA without introducing type of single molecule assay relying stability of the pRM repressible state any breaks in the DNA. We attach the on tethered particle motion (TPM) has at physiological CI levels and speculate PNA handles such that they flank the confirmed the sites in OL and OR nec- what features of supercoiling may be λ immunity region, tethering the DNA essary for clamping.9 Until recently, all responsible. molecule between a glass surface and a e27517-2 Bacteriophage Volume 4  Figure 2. (A) The size of the thermal fluctuations of the plasmid tethered bead as function of time in presence of 80 nM CI. The unlooped state causes the bead to exhibit larger fluctuations (indicated by the dotted line) than the looped state (dashed line). (B) Histogram corresponding to the time series shown in (A), the left peak corresponds to the looped state, the right peak to the unlooped state. (C) Chloroquine gel containing 4-fold dilutions of the supercoiled plasmid preparation. The large number of bands confirms that there is a large spread in supercoils (writhe number) in the sample. (D) Probability of CI-mediated looping as function of CI concentration for supercoiled DNA (red circles) published in reference 10 and for linear DNA (blue squared) published by Zurla et al.9 The lines show the corresponding thermodynamical models. Supercoiling enhances the binary response to changes in CI concentration and lowers the CI concentration necessary for pRM repression. The dashed box highlights the narrow sigmoidal transition interval for the supercoiled DNA. Interestingly, 20 nM CI (at the center of the transition interval) corresponds to 12 CI monomers available in the cell and this is exactly the number of CI molecules necessary to form a pRM repressible state. The number of data sets for each concentration is n = 4 for 5 nM CI, n = 7 for 20 nM CI, n = 5 for 40 nM CI, n = 6 for 80 nM CI, and n = 9 for 170 nM CI. Error bars represent one standard deviation. Effect of Supercoiling submicron sized polystyrene bead. TPM Brownian fluctuation (as routinely done on Looping Probability experiments confirmed that we could in the linear assays14) did not make sense distinguish the dynamics of supercoiled for the supercoiled assay. To overcome plasmids compared with that of relaxed Using the PNA-based single molecule this challenge, for each individual time plasmids and linear relaxed tethers.10 assay, the probability of a DNA plasmid series (as shown in Fig. 2A) we calculated Using this assay employing naturally being in the looped state is determined by the histogram (Fig. 2B) and assigned the supercoiled DNA, CI-mediated looping examining the thermal excursions of the lower peak to the looped state and the could be detected directly as a decrease tethered bead, higher peak to the unlooped state. This in the Brownian motion of the tethered assignment helped normalize all data sets bead, thus producing a telegraph-like sig- and collapse them into a single histogram. nal over time as shown in Figure 2A. We The ratio of the area under the peak rep- envision this PNA-based assay having resenting the looped state to the total area numerous applications beyond studies was then assigned as the probability of of the λ switch. It could enable studies as a function of time (see Fig. 2A). The looping. This procedure was performed of protein/enzyme DNA interactions on corresponding histogram is shown in at different CI concentrations, thus estab- supercoiled DNA mimicking the native Figure 2B where the peak centered lishing the looping probability as function bacterial DNA or eukaryotic chroma- around 180 nm corresponds to the looped of CI concentration for a supercoiled assay tin, without the requirement of external state and the peak around 250 nm to the as shown in Figure 2D (red circles). interference, e.g., magnetic tweezers.11-13 unlooped state. Finzi and coworkers examined We caution interested researchers that Plasmid DNA as recovered from CI-mediated looping probabilities using in our TPM studies of supercoiled DNA bacteria contains a varying number of a linear DNA assay in the physiologi- we took great care with the purity of all supercoils, the writhe number of any cally relevant CI concentration regime.9 reagents used: The plasmid DNA was single plasmid molecule being constant Their results are shown by blue squares in purified through two rounds of isopyc- throughout the looping experiments. To Figure 2D. In the linear assay, the DNA nic centrifugation with CsCl and ethid- examine the distribution in writhe num- looping probability curve can be described ium bromide, the PNA was purified by ber, we electrophoresed a sample through by a low Hill coefficient (h = 1.2). This HPLC and all solutions were filtered a chloroquine gel as shown in Figure 2C. means the repressible state of pRM is through several centimeter beds of fine The large variety in writhe between continually responsive to changes in CI sephadex to remove particles. We did plasmids with identical sequence and concentration and as CI is depleted with not examine reagents purified by other length posed a challenge to the interpre- activated RecA, pRM will compensate by methods and therefore cannot comment tation of the looping dynamics because trying to produce more CI. Considering on their suitability for the supercoiled the magnitude of the Brownian motion the sophistication of the prophage main- single molecule assay. We also manipu- from, say, an unlooped state would vary taining system, such counteraction of CI lated all solutions containing PNA/DNA from one tethered bead to another. Also, regulation against lytic induction would be with low-binding and DNAase free this meant that a calibration curve relat- surprising. We speculate that the observed plasticware. ing sequence length to magnitude of low Hill coefficient was due to the relaxed www.landesbioscience.com Bacteriophage e27517-3 nature of linear DNA. This is supported protein interactions. DNA supercoiling experiments lack parts of the machinery by the results utilizing the supercoiled has been found to facilitate protein-pro- of a living cell and hence some processes assay, where we found that the response tein and enhancer-promoter communica- may be more favorable in vivo, thus dis- of supercoiled DNA to varying concen- tion over large distances by increasing the playing a higher free energy. In compari- trations of CI was very different from the concentration of DNA segments in vicin- son to the parameters describing the linear response of linear or relaxed DNA10 (com- ity to each other.12,17,18 In addition, changes assay,9 we modeled the observed increased pare red circles to blue squares in Fig. 2D). in the degree of supercoiling induced by a cooperativity by lowering the intrinsic Instead of observing a continuous response previous RNA polymerase can either help binding energies between CI and each to CI concentration, the supercoiled assay a subsequent RNA polymerase to enter operator site by 1.5 kcal/mol, thus mak- exhibited a sharp transition between the directly into the open complex or impede ing multiple CI binding energetically more unlooped and looped state, the narrow the movement of a RNA polymerase.19 favorable than monomeric CI binding. In transition interval is highlighted by a Supercoiling is also known to facilitate total, with these parameters we thermody- dashed box in Figure 2D. At CI concen- unwinding of short DNA segments and namically allowed the biologically relevant trations above approximately 25% of the thereby expose single stranded DNA to octamer-mediated loop to be formed and average lysogenic concentration (~200 nM which regulatory proteins preferentially the negative total free energy change asso- CI15) the operators are nearly always in bind.13 Supercoiling is also thought to ciated with DNA looping, i.e., the sum of the looped, pRM-repressible state. Only affect gene regulation by changing the the two cooperative terms, indicated that when the CI concentration falls below local structure and dynamics of DNA 20 looping on supercoiled DNA is thermody- approximately 5% of the average lysogenic which can influence protein-DNA bind- namically favorable. concentration do the operators spend the ing by making the helical structure more majority of their time in the unlooped, or less accessible for the proteins. The pos- Supercoiling Tunes λ Immunity pRM-non-repressible state. sibilities are many and future investiga- The Hill coefficient describing the tions will be necessary to determine what Most interestingly, the minimum num- looping probability of naturally super- aspect of supercoiled DNA is responsible ber of CI molecules in the cell necessary coiled DNA as function of CI concen- for the higher Hill coefficient. to form the pRM-repressible state (12 CI tration was estimated to be h = 2.5 (in molecules) corresponds to a cellular con- contrast to h = 1.2 from the linear DNA). Thermodynamic Parameters centration of 20 nM. Our results on the Regulatory proteins rarely act alone but looping probability of supercoiled DNA instead cooperatively as a multi protein By analyzing our data in the light of (Fig. 2D) show that this minimum cellu- complex. A higher Hill coefficient can a thermodynamic model put forward in lar concentration corresponds to looping be interpreted as a higher degree of coop- refs.7,21,22 we estimated the cooperative the DNA between the operators 58% of erativity in a system. Cooperativity helps binding energies associated with loop for- time. Below 20 nM of CI, the probability ensure efficient discrimination between mation. The model including the free ener- of the operators being in the looped state two states and simultaneously enhances gies for the supercoiled system is shown as drops rapidly. Thus, during induction by a switch-like response to small changes in the red curve in Figure 2D. In the model, the SOS pathway, pRM remains in the regulator concentration. Recently it was ΔGoct represents the net free energy change repressible state until all of the free CI has shown in vivo that DNA looping could be due to octamerization of CI bound across been degraded with activated RecA. We abolished if CI cooperativity was elimi- OL-OR together with the cost of forma- propose that below this minimum thresh- nated, supporting the necessity of cooper- tion of a DNA loop, this was set to 0 kcal/ old, the CIs abruptly vacate OR and OL ativity for λ switch efficiency.16 Therefore, mol. ΔGtetr which represents the coopera- and lytic repression collapses. Despite the the observed increased Hill coefficient of tive free energy due to tetramerization of low copy number of CIs in the cell, the the supercoiled DNA is in agreement with CI bound across OL3-OR3 was set to -1.0 pRM repressible state is robust to both the binary developmental nature of the λ kcal/mol. By setting ΔGoct to 0 kcal/mol perturbations in gene expression that can switch. we require that supercoiling together with vary dramatically from cell to cell, and to octamerization of CI balances the cost of CI nonspecific DNA binding that would Features of Supercoiling Possibly bending the DNA into a loop. This is in otherwise lead to spontaneous transition Responsible for Higher Hill accordance with the facts that octamer- to lytic development. Based on our find- Coefficient mediated looping has been established to ings we propose that supercoiling plays be biologically relevant7 and that the bac- a role in stabilizing the pRM-repressible DNA supercoiling is an intrinsic prop- terial DNA has in vivo been shown to be state such that CI synthesis from pRM erty of the circular Escherichia coli DNA highly condensed even in the absence of does not impede induction. that helps compact the DNA in a highly nonspecifically bound CIs to help facilitate condensed form. Because of the topology the DNA looping.16 This value of ΔGoct is Concluding Remarks of supercoiled DNA it can in itself be con- also in agreement with in vivo observa- sidered as a regulatory parameter for many tions.7, 16 ΔGtetr differs somewhat from By using PNA as handles, we tethered cellular processes such as transcription and values estimated in vivo, however in vitro supercoiled DNA plasmids and utilized e27517-4 Bacteriophage Volume 4 the assay for a single molecule study of the efficient response to the cellular SOS sys- Acknowledgment λ switch in a naturally supercoiled sys- tem exactly at the cellular concentration We acknowledge financial support tem. This assay has potential to examine corresponding to the minimum number from the University of Copenhagen excel- how DNA in its natural supercoiled state of CI molecules capable of maintaining lence program. interacts with proteins, enzymes, histones the CI repressible state. in nucleosomes, and the transcription and replication machinery. Interestingly, Disclosure of Potential Conflicts of Interest our results show that the λ switch is No potential conflicts of interest were finely tuned to have an optimal, fast and disclosed. 10. Norregaard K, Andersson M, Sneppen K, Nielsen 17. Polikanov YS, Bondarenko VA, Tchernaenko V, References Jiang YI, Lutter LC, Vologodskii A, Studitsky VM. PE, Brown S, Oddershede LB. DNA supercoiling 1. Ptashne M. A Genetic Switch: Lambda Phage enhances cooperativity and efficiency of an epigenetic Probability of the site juxtaposition determines the Revisited. Cold Spring Harbor, NY: Cold Spring switch. 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