DNA binding properties of <i>Pf</i>-SSB^MDCC.

<p>(<b>A</b>) Purified <i>Pf</i>-SSB^MDCC was analyzed on a 12% SDS-PAGE gel and imaged after staining with coommassie dye or detected using fluorescence imaging. M denotes the protein ladder. (<b>B</b>) Excitation (blue, λ_ex) and emission (green, λ_em) spectra of 1 μM <i>Pf</i>-SSB^MDCC are shown. The dotted lines correspond to an excitation and emission maxima of 430 nm and 482 nm, respectively. (<b>C</b>) 1 μM <i>Pf</i>-SSB^MDCC was excited at 430 nm and emission spectra were measured in the absence of DNA (grey) and in the presence of a 125 bp dsDNA (blue) or an oligo-dT 70 nt ssDNA (red). A four-fold increase in <i>Pf</i>-SSB^MDCC fluorescence is observed in the presence of the (dT)₇₀ ssDNA oligonucleotide. (<b>D</b>) Fluorescence titration of <i>Pf</i>-SSB^MDCC with increasing concentrations of ssDNA [(dT)₇₀]. <i>Pf</i>-SSB^MDCC binds stoichiometrically, with one SSB tetramer binding to one (dT)₇₀ oligonucleotide as denoted by the dotted line. The mean values and standard errors from three independent experiments are shown. (<b>E</b>) Isothermal calorimetric measurement of changes in enthalpy associated with binding of two (dT)₃₅ molecules to <i>Pf</i>-SSB and <i>Pf</i>-SSB^MDCC are shown. Both proteins bind stoichiometrically with similar observed heat changes ΔH_<i>obs</i> = -73.1±0.2 kcal mol^-1 and -71.8±0.2 kcal mol^-1 for <i>Pf</i>-SSB and <i>Pf</i>-SSB^MDCC, respectively. The mean values and standard errors from three independent experiments are shown.</p

<i>Pf</i>-SSB^MDCC does not influence the activity of Srs2.

<p>(<b>A</b>) Change in fluorescence upon mixing varying concentrations of <i>Pf</i>-SSB^MDCC (100 or 200 nM) with buffer in the presence or absence of m13ssDNA (3 μM nucleotides). (<b>B</b>) Rad51 filament clearing by Srs2 was measured in the presence of increasing concentrations of <i>Pf</i>-SSB^MDCC. Preformed Rad51 filaments were rapidly mixed with varying amounts of <i>Pf</i>-SSB^MDCC (75, 100, 125 or 150 nM) and Srs2 (100 nm) and the change in fluorescence was measured over time. Data were collected over a split time period with 5000 points each assigned to the first 10 sec and remaining 50 sec, respectively. An average of 10 independent traces is shown. (<b>C</b>) The normalized change in fluorescence at time = 10 sec was subtracted from time = 0.01 sec and the Δfluorescence^@10sec values plotted as a function of [<i>Pf</i>-SSB^MDCC]. No significant change in fluorescence is observed. The mean values and standard errors from three independent experiments are shown.</p

2B domain mutations in Srs2 have no effect on its DNA unwinding capabilities.

<p>(<b>A</b>) Alignment of the region in the 2B domains from UvrD, Rep, PcrA and Srs2. D437 and K438 in Srs2 align with D403 and D404 in UvrD, which are mutated in the <i>uvrD303</i> phenotype, a hyperactive helicase mutant of UvrD. Amino acids are colored according to their physicochemical properties. (<b>B</b>) Crystal structure of the UvrD (PDB ID:2IS4; the bacterial Srs2 homolog) is shown with the 2B domain colored gold. The 2B domain is in the ‘closed conformation’ when bound to the unwinding DNA substrate. The DNA is shown as sticks (black) and the D403-D404 residues are shown as red spheres. (<b>C</b>) SDS-PAGE analysis of the purified full length Srs2^WT and Srs2^DK-AA proteins. (<b>D</b>) Unwinding kinetics of a DNA substrate (25bp dsDNA with a 16 nt 3' ssDNA overhang) by Srs2^WT and Srs2^DK-AA. No discernable difference in unwinding kinetics is observable between the two proteins. Fitting the linear portion of the data (insert) yield unwinding rates of 0.026 s^-1 and 0.028 s^-1 for the Srs2^WT and Srs2^DK-AA proteins, respectively. The mean values and standard errors from three independent experiments are shown.</p

<i>Pf</i>-SSB^MDCC binds stoichiometrically to ssDNA over a wide range of NaCl concentrations.

<p>(<b>A</b>) Fluorescence titration of <i>Pf</i>-SSB^MDCC with increasing concentrations of ssDNA [(dT)₇₀] in the presence of increasing concentrations of NaCl. Experiments were performed in 20 mM Tris-Cl, pH 8, 0.1 mM EDTA, and 1 mM TCEP with either 0.02 (●), 0.1 (■), 0.5 (○) or 1M (▲) NaCl in the reaction. (<b>B</b>) Stoichiometry of [<i>Pf</i>-SSB bound]/[(dT)₇₀] under various NaCl conditions from A is plotted as a function of [NaCl] and shows no significant change in binding stoichiometry over a wide range of NaCl concentrations. The mean values and standard errors from three independent experiments are shown. (<b>C</b>) Stopped-flow analysis of <i>Pf</i>-SSB^MDCC binding to (dT)₇₀ ssDNA. Rapid binding of <i>Pf</i>-SSB^MDCC to ssDNA is observed as increasing concentrations of (dT)₇₀ are mixed with a fixed concentration of <i>Pf</i>-SSB^MDCC (20 nM). Data were fit to a single exponential equation and (<b>D</b>) the k_obs (s^-1) from the fits were plotted as a function of DNA concentration yielding an apparent association rate constant 2.6 x 10⁸ M^-1s^-1.</p

Rad51 filament clearing by Srs2 captured by <i>Pf</i>-SSB^MDCC.

<p>(<b>A</b>) Stopped-flow measurement of <i>Pf</i>-SSB^MDCC (80 nM) binding to m13 circular ssDNA (3 μM nucleotides) is shown by a rapid increase in fluorescence (blue trace). No change in fluorescence is observed in the absence of ssDNA (black trace). (<b>B</b>) When m13ssDNA (3 μM nucleotides) is pre-coated with Rad51 (3 μM)in the presence of ATP (3 mM) and then mixed with <i>Pf</i>-SSB^MDCC (80 nM), no significant change in fluorescence is observed (green trace) suggesting that <i>Pf</i>-SSB^MDCC does not gain access to ssDNA when it is completely bound by Rad51 in the form of a nucleoprotein filament. (<b>C</b>) Challenging the Rad51 nucleoprotein filament on m13ssDNA with <i>Pf</i>-SSB^MDCC in the presence of full length Srs2 (25 nM) results in a gradual increase in the fluorescence signal (red). Srs2 clears the Rad51 from the ssDNA yielding free ssDNA for the rapid and tight binding of <i>Pf</i>-SSB^MDCC. Models for the reaction mixing schemes are presented above each data panel.</p

Domain organization of <i>Pf</i>-SSB.

<p>(<b>A</b>) Schematic representation of the DNA binding, protein-protein interaction and linker regions of <i>E</i>. <i>coli</i> and <i>P</i>. <i>falciparum</i> SSB. <i>Pf</i>-SSB also has an apicoplast localization signal (ALS), which is not required for its DNA binding function. The numbers denote positions of the amino acids at the beginning and end of each domain. (<b>B</b>) Individual subunits of the homotetrameric DNA binding domain are depicted as cartoon representation in the crystal structure of <i>Pf</i>-SSB. The Cys93 residues used for attachment of the fluorophore are shown as black spheres. (<b>C</b>) <i>Pf</i>-SSB is shown as surface representation with two (dT)₃₅ DNA molecules (blue stick representation) wrapped around the homotetramer. (<b>D</b>) The proximity of Cys93 (black stick) to the bound DNA in <i>Pf</i>-SSB is highlighted. (<b>E</b>) Structure of the MDCC (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)-carbonyl)coumarin) fluorophore used to label <i>Pf</i>-SSB. Images of the <i>Pf</i>-SSB structure were generated using PDB ID: 3ULP.</p

Nucleoprotein filament clearing activity of Srs2 is unaffected by mutations in its 2B domain.

<p>Stopped-flow analysis of Rad51 nucleoprotein filament clearing with increasing concentrations of (<b>A</b>) Srs2^WT and (<b>B</b>) Srs2^DK-AA were measured using <i>Pf</i>-SSB^MDCC as a reporter for free ssDNA in the reaction. (<b>C</b>) The percent change in <i>Pf</i>-SSB^MDCC fluorescence at time = 40 sec from A and B are plotted against Srs2 concentration and shows a hyperbolic relationship. The K_1/2 for Rad51 filament clearing was obtained by fitting the data to a hyperbola and shows that both proteins clear Rad51 filaments with similar efficiency. The mean values and standard errors from three independent experiments are shown.</p

Loss of homeostasis in small intestines of <i>vABKO a</i>nd <i>vTKO</i> mice.

<p>(<b>A</b>) Mice were injected with tamoxifen for five consecutive days and then sacrificed 3 days after the final injection. Small intestines were isolated and length determinations were made. Small intestine lengths were normalized to body weights, which were determined prior to the first tamoxifen-injection. Data is presented as mean +/− SEM. Asterisk (*) indicates significantly different after tamoxifen injection as determined by a Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. The actual P-values are 0.38 (WT), 0.76 (<i>vil-Cre-ER^T2</i>), 0.003 (<i>vAKO</i>), 0.31 (<i>vBKO</i>), 0.002 (<i>vABKO</i>), 0.46 (<i>vACKO</i>) and 0.004 (<i>vTKO</i>). The small intestinal lengths of <i>vAKO</i>, <i>vABKO</i> and <i>vTKO</i> mice were significantly different from WT mice injected with tamoxifen as determined by a Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. Actual P-values are 0.40 (<i>vil-Cre-ER^T2</i>), 0.03 (<i>vAKO</i>), 0.10 (<i>vBKO</i>), 0.00006 (<i>vABKO</i>), 0.36 (<i>vACKO</i>) and 0.0006 (<i>vTKO</i>). (<b>B</b>) Duodenums isolated from mice treated as described in A were photographed under a dissection microscope. Scale bar: 0.5 mm. (<b>C</b>) Significant shortening of villi in small intestines of <i>vABKO</i> and <i>vTKO</i> mice. Length of individual villi shown in panel B were measured (30 villi per mouse). Data is presented as mean +/− SEM. Asterisk (*) indicates significantly different after tamoxifen injection as determined by a Student's t-test. P-values are 0.00008 (<i>vABKO</i>) and 0.007 (<i>vTKO</i>). Villi lengths of <i>vil-Cre-ER^T2</i>, <i>vABKO</i> and <i>vTKO</i> mice were significantly different from WT mice injected with tamoxifen as determined by a Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. Actual P-values are 0.001 (<i>vil-Cre-ER^T2</i>), 0.27 (<i>vAKO</i>), 0.45 (<i>vBKO</i>), 0.0005 (<i>vABKO</i>), 0.25 (<i>ACKO</i>) and 0.03 (<i>vTKO</i>).</p

Targeted disruption of <i>Cdc25B</i> in mice.

<p>(<b>A</b>) Structure of targeting vector and chromosomal organization of <i>Cdc25B</i> locus before and after Cre-mediated excision. The genomic organization of the mouse <i>Cdc25B</i> gene was disrupted by inserting into intron 1 the neomycin phosphotransferase cDNA driven by the phosphoglycerine kinase promoter (pGK-neo) as a selectable marker. Exons are represented by black boxes. The location of Hind III (H), Bam HI (B) and Kpn I (K) site is indicated and <i>loxP</i> sites are represented by yellow triangles. Sizes of upstream (3.3 kb) and downstream (4.5 kb) homologous arms are indicated. Position of probes used for Southern blotting are shown. Red triangles depict the locations of PCR primers used for genotyping. Abbreviations: +, wild type allele; R, recombinant allele; F, floxed allele; WT, wild type. (<b>B</b>–<b>C</b>) Southern blot analysis demonstrating homologous recombination in the <i>Cdc25B</i> locus. ES cell genomic DNA was digested with Hind III (B) and Bam HI (C), and Southern blotting was performed using the 5′ and 3′ probes shown in panel A. The genotype of each ES cell line is indicated. The location of size markers is shown on the left. (<b>D</b>) Southern blot analysis demonstrating Cre-mediated recombination in the <i>Cdc25B</i> locus. ES cell clones containing the recombinant allele were expanded and transiently transfected with a plasmid encoding Cre recombinase. Genomic DNA was digested with Kpn I (K), and Southern blotting was performed using the internal probe shown in panel A. The genotype of each ES cell line is indicated. Location of size markers is shown on left. (<b>E</b>) PCR analysis of mouse tail DNA. Mouse tail DNA was amplified with PCR primers depicted as red triangles in panel A. The wild type (+) allele produced a 383 bp PCR product and floxed allele (F) produced a 433 bp PCR product. The genotype of each mouse is indicated. The location of size markers is shown on the right.</p

Tamoxifen injection induced efficient recombination within <i>Cdc25A</i> and <i>Cdc25B</i> loci.

<p>(<b>A</b>) Genomic DNA isolated from the small and large intestines of tamoxifen-treated mice were assessed for Cre-mediated excision by Southern blotting. Deletion frequencies are shown below each lane and were determined by measuring band intensities using a Molecular Dynamics Storm imager. (B) Total RNA isolated from the small intestine (jejunum) of tamoxifen-treated WT and <i>vB^f/−</i> mice was reverse-transcribed into cDNA. qRT-PCR was used to determine relative amounts of <i>Cdc25B</i> mRNA. The data is presented as mean +/− SEM. Asterisk (*) indicates significantly different from WT, P  =  0.012 by Student's t-test. Note that <i>vB^f/−</i> mice are generated by crossing <i>Cdc25B</i> null mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015561#pone.0015561-Lincoln1" target="_blank">[26]</a> with <i>Cdc25B</i> conditional mice. The PCR primers detect transcript arising from the null allele but not the deleted floxed allele. Thus, a 50% decrease in relative expression indicates complete loss of <i>Cdc25B</i> expression.</p