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