NLS of SA1 identified in yeast is the same in HeLa cells but SA2 is targeted to the nucleus of HeLa by C-terminally localized NLS.

<p>(A) Schematic representation of HeLa cells expressing SA1-GFP and SA2-GFP and their deletion mutants. Arrows indicate localization of NLS discussed in the text. Grey color indicates GFP fluorescence. (B) HeLa cells expressing SA1-, SA2L- and SA2S-GFP fusion proteins. (C) HeLa cells expressing SA1-GFP and SA2L-GFP devoid of N-terminal NLS 34–53 and 32–47, respectively. (D) HeLa cells expressing SA2L protein devoid of 161 C-terminal amino acids (upper panel), C-terminal NLS 1071–1140 (middle panel), C-terminal NLS 1199–1206 (lower panel). GFP represents fluorescence of fusion proteins, DNA was stained with DAPI.</p

NLS of H2B does not confer nuclear localization on SA2S.

<p>(A) Yeast cells expressing fusion protein H2B^1–62-SA2S-GFP. (B) H2B^1–62-SA2S-GFP protein has predicted molecular weight. Diploid yeast strain <i>irr1Δ</i>/<i>IRR1</i> (lacking one copy of <i>IRR1</i> gene) was transformed with centromeric plasmid pUG35 bearing hybrid gene encoding the fusion protein. Details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038740#pone-0038740-g001" target="_blank">Figure 1</a>.</p

Plasmids used in this study.

<p>Abbreviations for description of plasmids: CEN, centromeric; 2 µ, episomal; MCS, multiple cloning site.</p

SA2S contains NES functional in yeast between L953 and M962.

<p>Consensus for Crm1p-dependent export: ΦX_2–3ΦX_2–3ΦXΦ, where Φ represents L, I, V, F or M and X – any amino acid. (A) Left – cells expressing fusion protein SA2S-GFP. Right – cells expressing SA2SF960E–GFP protein bearing the substitution F960E which disrupts NES 953 LEK FMT <u>F</u>QM 962. The SA2SF960E–GFP protein accumulates in the nucleus in 100% of cells. DNA was stained with DAPI, GFP represents fluorescence of fusion proteins, VIS – transmitted light. (B) SA2S-GFP protein with NES signals disrupted by site-directed mutagenesis. SA2V699S – inactivated NES between positions 689 and 699, SA2SF960E – between positions 953–964. At least 100 cells were counted. Bars show percentage of cells with a given localization of GFP signal. Protein localized to the cytoplasm – black, to the nucleus–grey.</p

Pre-steady state kinetics of correct nucleotide incorporation by the Pol^D714AExo- mutant.

<p>Reactions to measure the incorporation of dATP opposite template T by RB69 DNA Pols were carried out at 10°C. A pre-incubated solution containing the enzyme (1 µM) and radiolabeled 13*/19-mer DNA substrate (100 nM) was mixed with 10mM MgSO₄ and dATP (0.001 mM – 1.5 mM). The reactions were quenched by addition of 0.5 M EDTA (pH 8.0) and analyzed on denaturing polyacrylamide gels. The data were fit to a single-exponential equation to obtain k_<i>obs</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076700#pone.0076700.s002" target="_blank">Figure S2</a>). These values were subsequently plotted as a function of dATP concentration for D714A and wild type RB69 DNA Pols and k_<i>pol</i> and K_{<i>d,dNTP</i>} were calculated as described in Materials and Methods. The standard deviations (SD) are shown as error bars (<b>A</b>) and ± values (<b>B</b>). A close-up of the mutant plot is depicted on the right. The catalytic efficiency of each polymerase was obtained by dividing its respective k_<i>pol</i> by K_{<i>d,dNTP</i>} (<b>B</b>).</p

Crystal structures of a catalytic complex of the Pol^Y567A/D714A mutant.

<p>(<b>A</b>) The active site of the mutant in ternary complex I. The end of the primer is shown with incoming dGpnpp (magenta), and bound metal ions: sodium (purple sphere), calcium (yellow sphere), and coordinating waters (red spheres). A simulated-annealing <i>F</i>_o-<i>F</i>_c omit map, contoured at 3σ, is shown in blue. (<b>B</b>) Overlay of ternary complex I (PDB ID code: 4I9Q) and the wild type (PDB ID code: 3NCI) polymerase active sites in their respective ternary complexes highlighting the different conformation of the phosphates of the incoming nucleotide, the metal ions and the flip of the 411 side chain. The D714 mutant structure is rendered with the same colors as in (<b>A</b>). The wild type structure is rendered in gray. The α-, β-, and γ-phosphates of the incoming nucleotide are marked with arrows. <b>C</b>) A network of interactions propagates the perturbation resulting from the absence of the 714 side chain into the active site, influencing the conformation of D411. The color scheme is as in (<b>B</b>). (<b>D</b>) The active site in ternary complex II. The DNA (yellow), incoming dGpnpp (cyan) and a calcium ion (yellow sphere) are shown. A simulated-annealing <i>F</i>_o-<i>F</i>_c omit map, contoured at 4σ, is shown in blue. (<b>E</b>) Overlay of ternary complex II (PDB ID code: 4KHN) and the wild type (PDB ID code: 3NCI) polymerase active sites, highlighting the large differences in the conformation of the β- and γ-phosphates of the incoming nucleotide. Like for ternary complex I, a flip in the D411 side chain can be observed relative to the wild-type structure. The α-, β-, and γ-phosphates of the incoming nucleotide are marked with arrows. (<b>F</b>) The absence of the 714 side chain in ternary complex II results in similar structural perturbations to those observed in ternary complex I (see C). Due to the lack of the electron density the side chain of E716 was not shown.</p

DNA binding affinity and exonuclease activity of Pol^D714A on dsDNA.

<p>DNA binding affinities of the wild type RB69 and Pol^D714A polymerases (<b>A</b>), as well as their exonuclease deficient derivatives (<b>B</b>), were determined by DNA mobility-shift assays. A radiolabeled 20*/26-mer primer-template DNA substrate was incubated with increasing amounts of each polymerase, and the resulting DNA-protein complexes were analyzed on native 6% polyacrylamide gels (<b>A</b> and <b>B</b>). The amount of bound DNA substrate was quantified as an average from two independent experiments and plotted against protein concentration (<b>C</b> and <b>D</b>). (<b>E</b>) K_<i>Dapp</i> was calculated using Kaleidagraph (Synergy Software). The additional shifted DNA species visible for Exo⁺ variants at higher protein concentrations may correspond to more than one molecule of polymerase bound per oligonucleotide substrate. (<b>F</b>) A ³²P-labeled 20*/26-mer DNA substrate was incubated with the mutant or wild-type RB69 DNA Pol for 10, 20, 40, 80, 180 and 300 sec. at 37°C. Products of DNA degradation were analyzed on denaturing polyacrylamide gels and visualized on a phosphorimager. (<b>G</b>) The amount of undigested DNA substrate was calculated and plotted as a function of time. Data are averages from three independent experiments for each polymerase. Pol^D714AExo⁺ displays slightly elevated exonuclease activity, reflected in an only ~ 10% increase in DNA substrate consumption at the shortest incubation times. The observed difference between the exonucleolytic activities of the enzymes appears therefore to be negligible.</p

Crystal structure of the Pol^D714A mutant at 2.60 Å resolution.

<p>(<b>A</b>) Overlay of the wild type (PDB ID code 1IH7; navy blue) and Pol^D714A apoenzyme structures (PDB ID code: 4I9L; yellow), showing the overall fold of the mutant enzyme. The positions of the D714 and D411 residues of the wild type RB69 Pol are indicated. Polymerase subdomains are shown as transparent surfaces in different colors. (<b>B</b>) Simulated annealing omit map contoured at 1.5σ, showing the region surrounding D714 in the Pol^D714A crystal structure (yellow), overlaid with the corresponding region of the wild type RB69 Pol structure (cyan). D714 of RB69 Pol is colored magenta; hydrogen bonds between interacting residues are shown as dashed lines.</p

SA2S shuttles between nucleus and cytoplasm in yeast cells.

<p>(A) Subcellular localization of SA2S-GFP was analyzed after addition of LMB (Crm1p inhibitor) to 40 ng/ml to cells in logarithmic phase of growth. Strain <i>CRM1-T539C</i> bears LMB-sensitive version of Crm1p. Fourth column shows a composite of two fields from a single experiment but photographed as separate images, as marked. (B) Localization of SA2S-GFP protein was analyzed in thermo-sensitive <i>crm1-1</i> mutant. Transfer of cells grown at 30°C to 37°C for 30 minutes caused nuclear shift of the fusion protein in 100% of cells. Third and fourth columns show a composite of two fields from a single experiment, as marked. On the right in (A) and (B) control experiments in wild-type yeast are shown. DNA was stained with DAPI, GFP represents fluorescence of fusion proteins, VIS – transmitted light. (C) Frequencies of cells localized predominantly to the cytoplasm (black) or to the nucleus (gray) in strains bearing <i>CRM1-T539C</i> (LMB-sensitive) or <i>crm1-1</i> (thermo-sensitive) versions of Crm1p, following LMB treatment or temperature shift, respectively. MN47 and ABL10 are corresponding control strains bearing wild type <i>CRM1</i> gene, subjected to the same treatments.</p

SA1 contains NLS functional in yeast between 34K and 53K.

<p>(A) – Cells expressing SA1Δ34–53-GFP. (B) – Cells expressing fusion protein SA1^1–71-GFP. DNA was stained with DAPI, GFP represents fluorescence of fusion proteins, VIS – transmitted light. Column (A) shows a composite of two fields from a single experiment but photographed as separate images, as marked. For subcellular localization of intact.</p