Dependence of the length of the RecA–DNA filament on the applied stretching force for torsionally constrained DNA

<p><b>Copyright information:</b></p><p>Taken from "Torque-limited RecA polymerization on dsDNA"</p><p>Nucleic Acids Research 2005;33(7):2099-2105.</p><p>Published online 11 Apr 2005</p><p>PMCID:PMC1075924.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Data are taken at the end of the filament formation when the assembly reaction has stalled. Up to 3.6 pN, the length of the RecA–DNA filament increases (circles). Above 3.6 pN, the size of the RecA–DNA filament remains constant (diamonds). Solid line is the outcome of the model described in ‘Discussion’. Note that the model curve is plotted without any adjustable parameters. Above 3.6 pN, the model does not apply because the assembly reaction is not stalled by the formation of plectonemes but by a built-up torsion in the remaining DNA molecule

On torsionally constrained dsDNA, RecA–DNA filament formation stalls at a certain length that is dependent on the applied stretching force

<p><b>Copyright information:</b></p><p>Taken from "Torque-limited RecA polymerization on dsDNA"</p><p>Nucleic Acids Research 2005;33(7):2099-2105.</p><p>Published online 11 Apr 2005</p><p>PMCID:PMC1075924.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> This is due to the formation of positive plectonemes in the remaining uncovered DNA. Removal of these positive plectonemes in the remaining DNA molecule by rotation of the external magnets allows the assembly reaction of a RecA–DNA filament to continue. The molecule reached its maximal extension after 355 ± 10 and 320 ± 10 negative turns by the magnets at a stretching force of 1.8 and 0.4 pN, respectively. From force-extension measurements in both the cases, an effective length increase of 51 ± 2% is obtained compared with the DNA contour length

() Typical IHF–DNA complexes imaged as described in Materials and Methods

<p><b>Copyright information:</b></p><p>Taken from "Analysis of scanning force microscopy images of protein-induced DNA bending using simulations"</p><p>Nucleic Acids Research 2005;33(7):e68-e68.</p><p>Published online 20 Apr 2005</p><p>PMCID:PMC1083423.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The protein-induced bend is indicated by the arrows. Owing to its size, the IHF protein cannot be unambiguously identified in the images, demonstrating the need for an analytical approach using other than visual characteristics. The scale bar is 50 nm. Gray scale represents height ranging from 0 nm (dark) to 2 nm (bright). () Distributions of EED values normalized by contour lengths of IHF–DNA complexes (top) and bare DNA molecules (bottom), demonstrating the effect of DNA bending. () Histogram of bending angles estimated using tangents from IHF–DNA complexes. The bimodal distribution shows that not all DNA molecules have IHF bound; by fitting to a double Gaussian distribution, we estimate that ∼50% of the imaged molecules have IHF bound

(–) Histograms of experimentally obtained values for EED normalized by contour lengths of bare DNA molecules and protein–DNA complexes and the corresponding fits

<p><b>Copyright information:</b></p><p>Taken from "Analysis of scanning force microscopy images of protein-induced DNA bending using simulations"</p><p>Nucleic Acids Research 2005;33(7):e68-e68.</p><p>Published online 20 Apr 2005</p><p>PMCID:PMC1083423.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> (–) χ profiles for the data sets (solid lines with squares). The intersections with the dashed line indicate the uncertainty in the angle determination ()

Mms21-dependent sumoylation requires an intact Smc5/6 complex.

<p><b>A</b>. Composition of Smc5/6, depicting the different entities present in the complex. Nse subunits are labeled 1 to 6; Nse2 = Mms21. <b>B</b>. Sumoylation of Smc5 in <i>smc6</i> mutant cells. Samples of wild type and <i>GAL-3HA-SMC6</i> were collected from cells growing exponentially in galactose (<i>GALp</i> ON), or 12 h after shift to glucose to repress <i>3HA-SMC6</i> expression (<i>GALp</i> OFF). A <i>GALp-3HA-SMC6</i> strain expressing the <i>smc6-1</i> allele from a centromeric vector was also included in the analysis. Protein extracts were processed for HF-SUMO pull down as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002089#pbio.1002089.g001" target="_blank">Fig. 1G</a>. <b>C</b>. Co-immunoprecipitation analysis of the Smc5-Mms21 interaction from wild type and <i>smc6-1</i> protein extracts. Wild type and <i>GALp-SMC6 smc6-1</i> cells expressing Smc5-9myc and Mms21-6HA were shifted to glucose for 12 h and processed for anti-HA immunoprecipitation. <b>D</b>. Chromatin fractionation assay from wild type and <i>smc6-1</i> cells to analyze the amount of chromatin-bound Mms21-6HA. Controls for a chromatin-bound protein (histone H3) and cytoplasmic soluble (Hexokinase; Hxk) proteins are shown. <b>E</b>. Temperature and methyl methanesulfonate (MMS)-sensitivity of <i>nse</i> hypomorphic alleles. Growth test of wild type, <i>nse3-2</i>, and <i>nse5-2</i> cells in YPD plates at 25°C (containing or not the indicated MMS concentration) or at 37°C. <b>F</b>. Analysis of the Smc5-Nse3 and Smc5-Nse5 interaction in <i>nse</i> hypomorphic alleles. Exponentially growing Smc5-6Flag cells, expressing 9myc-tagged versions of either the wild type or the indicated hypomorphic <i>nse</i> alleles, were shifted to 37°C for 2 h (37) or kept at 25°C (25) before Smc5-6Flag immunoprecipitation. <b>G</b>. Co-immunoprecipitation analysis of the Smc5-Mms21 interaction in <i>nse3-2</i> and <i>nse5-2</i> mutant cells. Smc5-6Flag was immunoprecipitated, as in F, from cells grown at the indicated temperatures. Co-immunoprecipitation of Mms21-6HA was analyzed by western blot. <b>H</b>. Chromatin fractionation assay from Mms21-6HA tagged wild type and <i>nse5-2</i> cells, as in D. <b>I</b>. HF-SUMO pull down from wild type, <i>nse3-2</i>, or <i>nse5-2</i> cells expressing Smc5-6HA, before and after a shift to 37°C. In B and G, arrow points to unmodified form of the proteins, vertical bar to sumoylated forms. In D and H, WCE: Whole Cell Extract; SN: Supernatant; Chr: Chromatin fraction.</p

Up-regulation of Mms21-dependent sumoylation through expression of an E3-E2 fusion suppresses the smc5-DLEL coiled coil mutant.

<p><b>A</b>. HF-SUMO pull-down analysis from wild-type or E3-E2 cells, expressing 9myc-tagged wild-type or <i>DLEL</i> mutant versions of the Smc5 protein form its endogenous location, as indicated. <b>B</b>. Growth test analysis of wild type, <i>E3-E2</i>, <i>smc5-DLEL</i>, and double <i>E3-E2 smc5-DLEL</i> mutant cells; plates were incubated at 30°C in the presence or absence of 0.01% MMS. In A, arrow points to unmodified Smc5; vertical bars are sumoylated forms.</p

The coiled coil domain of Smc5 participates in activation of the Mms21 SUMO ligase.

<p><b>A</b>. Coiled coil probability of the Smc5 protein sequence in different species (<i>Saccharomyces cerevisiae</i>, <i>Ashbya gossypii</i>, <i>Magnaporthe grisea</i>, <i>Kluyveromyces lactis</i>, <i>Schizosaccharomyces pombe</i>, <i>Arabidopsis thaliana</i>, <i>Oryza sativa</i>, <i>Drosophila melanogaster</i>, <i>Danio rerio</i>, <i>Xenopus laevis</i>, <i>Gallus gallus</i>, <i>Mus musculus</i>, <i>and Homo sapiens)</i>; sequences are aligned according to P393 position in budding yeast. Numerical values for coiled coil probability are colored as shown in the legend; small vertical lines mark position of proline residues, inverted arrowheads mark position of proline residues in coiled coils. <b>B</b>. HF-SUMO pull-down analysis from wild-type cells expressing the indicated <i>SMC5-9myc</i> alleles from a centromeric plasmid; <i>DLEL</i> mutant contains the H391D, P393E, and E394L mutations. <b>C</b>. Co-immunoprecipitation analysis of the Smc5-Mms21 interaction in wild-type and <i>smc5-DLEL</i> mutant cells. <i>GALp-SMC5</i> cells expressing wild-type <i>SMC5</i> or <i>smc5-DLEL</i> allele from a centromeric vector were shifted to glucose for 6 h before collection. Mms21-6HA was immunoprecipitated from protein extracts (input) with anti-HA beads (IP); samples were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. <b>D</b>. Growth test analysis of <i>GALp-SMC5</i> cells transformed with the indicated plasmids and plated in glucose-containing media at 30°C in the presence or absence of MMS 0.01%. <b>E</b>. Nuclear segregation defects in <i>smc5-DLEL</i> cells after DNA damage. Wild-type and <i>smc5-DLEL</i> cells were arrested in G1 with alpha factor, treated with MMS 0.01% for 30 min, and released into the cell cycle; samples were taken at the indicated times for microscopic analysis, as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002089#pbio.1002089.g001" target="_blank">Fig. 1E</a>. <b>F</b>. HF-SUMO pull-down analysis in <i>GALp-SMC5 SMC1-6HA</i> cells expressing the indicated <i>SMC5-9myc</i> alleles from a centromeric vector; cells were shifted from galactose to glucose 6 h before collection to switch off the <i>GAL</i> promoter. In B and F, arrow points to unmodified SMC proteins; vertical bars are sumoylated forms.</p

The Smc5-Mms21 interaction is required for sumoylation of Mms21 targets and chromosome segregation after DNA damage.

<p><b>A</b>. Models for Mms21-dependent sumoylation: Mms21 may target proteins (including cohesin subunits) from its location in the Smc5/6 complex (left), or independently from Smc5/6 (right). <b>B</b>. Scheme of Mms21-binding surface on the coiled coil 2 of the Smc5 protein and location of mutated sites. <b>C</b>. Co-immunoprecipitation analysis of the Smc5-Mms21 and Smc5-Nse4 interactions. <i>MMS21-6HA</i> and <i>NSE4-6HA</i> cells were transformed with centromeric plasmids expressing the indicated <i>SMC5</i> alleles and subjected to anti-HA immunoprecipitation. <b>D</b>. Growth test analysis of <i>GALp-SMC5</i> cells transformed with the indicated centromeric plasmids. <b>E</b> and <b>F</b>. <i>GALp-SMC5</i> cells bearing the indicated vectors were shifted to glucose for 4 h to repress expression of the endogenous <i>SMC5</i> gene, and then treated as depicted in the figure; samples were taken at the indicated times for 4’,6-diamidino-2-phenylindole (DAPI) staining and microscopic examination (E) or Fluorescence-Activated Cell Sorting (FACS) analysis (F). Rectangles in F mark cells with less than 1N DNA content. <b>G</b>. <i>GALp-SMC5</i> cells ectopically expressing the indicated <i>SMC5</i> alleles from a centromeric vector were shifted to glucose for 6 h; 6xHis-Flag (HF) tagged SUMO was pulled down (P.D.) under denaturing conditions from yeast protein extracts (Input) to purify sumoylated species. Input and P.D. samples were analyzed by western blot with the indicated antibodies. <b>H</b>. <i>GALp-SMC5 SMC1-6HA</i> cells expressing the indicated <i>SMC5</i> alleles from a plasmid were shifted to glucose for 6 h. Protein extracts were processed for SUMO pull-down analysis as in G to analyze Smc1 sumoylation. In C, G, and H: wt = wild type, S1 = <i>smc5-S1</i>, S2 = <i>smc5-S2</i>, S3 = <i>smc5-S3</i>. In G and H, arrow points to unmodified form of the proteins, vertical bar to sumoylated forms; re-probing with anti-Flag is shown as a loading control for total SUMO in the purification.</p

ATPase-dependent activity of the Mms21 SUMO ligase.

<p><b>A</b>. Growth test of <i>GALp-SMC5</i> cells expressing wild-type <i>SMC5</i>, <i>smc5(K75I)</i>, or <i>smc5(D1014A)</i> from a centromeric vector in plates containing galactose (<i>GALp</i> ON) or glucose (<i>GALp</i> OFF). <b>B</b>. Mms21-3HA was immunoprecipitated from exponentially growing cells transformed with the indicated <i>SMC5</i>-expressing centromeric plasmids to test the Smc5-Mms21 interaction; wt = wild type; KI = <i>smc5(K75I)</i>; DA = <i>smc5(D1014A)</i>. <b>C</b>. Sumoylation analysis of ATPase-defective Smc5-9myc proteins. HF-SUMO pull-down analysis in wild-type cells transformed with plasmids expressing the indicated <i>SMC5</i> alleles. <b>D</b>. Sumoylation analysis of Nse4-6HA in <i>smc5</i> ATPase mutant cells. HF-SUMO pull-down analysis in <i>GALp-SMC5 NSE4-6HA</i> cells expressing the indicated <i>SMC5</i> alleles. Cells were shifted to glucose 6 h before collection to repress the endogenous <i>SMC5</i> gene. <b>E</b>. Sumoylation analysis of cohesin in <i>smc5</i> ATPase mutant cells. HF-SUMO pull down from cells of the indicated genotype (wt, <i>mms21ΔC</i> and <i>GALp-SMC5</i>), carrying a C-terminal 6HA tag on <i>SMC1</i>, and expressing or not an ectopic copy of <i>SMC5-9myc</i> (WT) or <i>smc5(K75I)-9myc</i> (KI) allele; where indicated, cells were treated with MMS 0,02% for 1 h (MMS) before collection. <b>F</b>. Chromatin fractionation assay from <i>GALp-SMC5 MMS21-6HA</i> cells expressing an ectopic 9myc-tagged copy of the indicated <i>SMC5</i> alleles, collected 6 h after shift to glucose to deplete the endogenous Smc5 protein. Controls for a chromatin-bound protein (histone H3), nuclear soluble (Rpd3) and cytoplasmic soluble (Hexokinase; Hxk) proteins are shown; WCE: Whole Cell Extract; SN: Supernatant; Chr: Chromatin fraction. In C–E, arrow points to unmodified Smc5, Nse4, or Smc1 proteins, and vertical bars to their sumoylated forms.</p

Binding of ATP to the ATPase head of Smc5 stimulates sumoylation in vitro.

<p><b>A</b>. Experimental outline for the purification of wild-type or K75I mutant Smc5/6-Mms21 complexes used in the reactions. <b>B</b>. In vitro sumoylation reactions on immunoprecipitated Smc5-9myc. Reactions were stopped after 1 h of incubation at 37°C with the human E1, E2, and SUMO enzymes, as described in Materials and Methods, and analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. <b>C</b>. Quantification of in vitro sumoylation rate in immunoprecipitated Smc5/6-Mms21 complexes, as described in Materials and Methods. Graph shows mean ± s.e.m.; <i>n</i> = 4; for each individual experiment, the rate of sumoylation for wild—type Smc5 was set to 1. <b>D</b>. In vitro sumoylation assay of the c-terminal domain (ct) of Nse4 (residues 246 to 402), using the Smc5-Mms21 heterodimer as the E3. Reactions were initiated by addition of ATP (time 0) and stopped at the indicated times. Samples were loaded in SDS-PAGE gels and stained with SYPRO-Ruby. <b>E</b>. Quantification of Nse4(ct) sumoylation rates, as described in Materials and Methods. Graph shows mean ± s.e.m.; <i>n</i> = 4; for each experiment, the rate of sumoylation using wild-type Smc5 was set to 1. wt = wild type; KI = <i>smc5(K75I)</i>. In B, asterisk marks unspecific band detected by the anti-SUMO2/3 antibody in immunoprecipitates.</p