Hybridization of the Sequoia complex with the DNA origami scaffold.

<p>(A) Schematic (top left) and negative stained EM of the Sequoia complex (bottom left, scale bar-20nm), and the proposed reconstitution of the in vivo Ndc80 complex architecture using the Sequoia complex and the 6-site origami scaffold (right). (B) Fluorescence intensity and the deduced number of Cy3 molecules per scaffold (n ≥ 99). (C) Mobility (top) and intensity analysis (bottom) of the scaffolds hybridized with the Sequoia complex (n = 3).</p

Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds

<div><p>DNA origami provides a versatile platform for conducting ‘architecture-function’ analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis.</p></div

Using DNA origami scaffolds for the architecture-function analysis of the yeast kinetochore.

<p>(A) <i>In vivo</i> architecture of the yeast kinetochore-microtubule attachment (left), and schematic of the desired in vitro reconstitution and alteration of this architecture (right). (B) 2-D class average of the EM image (left, scale bar- 50 nm), and salient features of origami (right). (C) Scatter plot of intensities for Single-particle TIRF measurements (n>101, horizontal line represents the mean and the vertical line s.e.m in this and all the scatter plots that follow). The corresponding number of Cy3 molecules displayed on the right y-axis. (D) Unimodal Cy5 intensity distribution reveals that the analyzed scaffolds were monomeric.</p

Exploiting GST dimerization for maximizing hybridization efficiency.

<p>(A) Schematic of dimeric (left) and monomeric (right) Sequoia hybridized to origami scaffold. (B) Single particle Cy3 fluorescence and the number of molecules for dimeric Sequoia hybridization before (left, n≥117) and after GST removal (right, n≥92). (C) The number of Sequoia molecules per scaffold from B (mean ± s.e.m). (D) Mobility of scaffolds conjugated with dimeric and monomeric Sequoia molecules (top) and Cy3 intensity quantitation (bottom, n = 3).</p

Cohesin Rings Devoid of Scc3 and Pds5 Maintain Their Stable Association with the DNA

<div><p>Cohesin is a protein complex that forms a ring around sister chromatids thus holding them together. The ring is composed of three proteins: Smc1, Smc3 and Scc1. The roles of three additional proteins that associate with the ring, Scc3, Pds5 and Wpl1, are not well understood. It has been proposed that these three factors form a complex that stabilizes the ring and prevents it from opening. This activity promotes sister chromatid cohesion but at the same time poses an obstacle for the initial entrapment of sister DNAs. This hindrance to cohesion establishment is overcome during DNA replication via acetylation of the Smc3 subunit by the Eco1 acetyltransferase. However, the full mechanistic consequences of Smc3 acetylation remain unknown. In the current work, we test the requirement of Scc3 and Pds5 for the stable association of cohesin with DNA. We investigated the consequences of Scc3 and Pds5 depletion <em>in vivo</em> using degron tagging in budding yeast. The previously described DHFR–based N-terminal degron as well as a novel Eco1-derived C-terminal degron were employed in our study. Scc3 and Pds5 associate with cohesin complexes independently of each other and require the Scc1 “core” subunit for their association with chromosomes. Contrary to previous data for Scc1 downregulation, depletion of either Scc3 or Pds5 had a strong effect on sister chromatid cohesion but not on cohesin binding to DNA. Quantity, stability and genome-wide distribution of cohesin complexes remained mostly unchanged after the depletion of Scc3 and Pds5. Our findings are inconsistent with a previously proposed model that Scc3 and Pds5 are cohesin maintenance factors required for cohesin ring stability or for maintaining its association with DNA. We propose that Scc3 and Pds5 specifically function during cohesion establishment in S phase.</p> </div

Depletion of Scc3 and Pds5 does not affect cohesin association with chromatin.

<p>Yeast strains were staged in G1 with <i>α</i>-factor and released into media with nocodazole. Chromosomal spreads were prepared as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002856#pgen-1002856-g002" target="_blank">Figure 2</a>. At every time point fluorescence of 50 nuclei was determined. Error bars represent standard deviation. FACS analysis of cellular DNA content is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002856#pgen.1002856.s013" target="_blank">Figure S13</a>. The strains were in (A): 1813 (<i>SCC1-Myc18</i>, <i>SCC3-HA6</i>), 1625 (<i>SCC1-Myc18</i>, <i>SCC3-HA6</i>-degron), 1815 (<i>SCC1-Myc18</i>, <i>PDS5-HA6</i>), 1818 (<i>SCC1-Myc18</i>, <i>PDS5-HA6</i>-degron), in (B): 1771 (<i>SCC3-Myc18</i>, <i>PDS5-HA6</i>), 1796 (<i>SCC3-Myc18</i>, <i>PDS5-HA6</i>-degron), 1734 (<i>PDS5-MYC18</i>, <i>SCC3-HA6</i>) and 1744 (<i>PDS5-Myc18</i>, <i>SCC3-HA6</i>-degron) in (C): 1479 (<i>SCC3-HA6</i>), 1864 (<i>SCC3-HA6, Δwpl1</i>), 1677 (<i>PDS5-HA6</i>), 1866 (<i>PDS5-HA6, Δwpl1</i>), 10589 (<i>SCC1-Myc18</i>) and 1906 (<i>SCC1-Myc18, Δwpl1</i>).</p

Depletion of Scc3 and Pds5 with a “conventional” temperature-sensitive degron.

<p>(A–C) Strains 2395 (<i>SCC1-HA6</i>), 2452 (<i>SCC1-HA6</i>, degron-<i>MYC18-PDS5</i>), 2455 (<i>SCC1-HA6</i>, degron-<i>MYC18</i>-<i>SCC3</i>) and 2456 (<i>SCC1-HA6</i>, degron-<i>MYC18- PDS5, degron-MYC18-SCC3</i>) were arrested with nocodazole in YEP raffinose at 30°C for 2 hours, resuspended in YEP galactose containing nocodazole and incubated for 45 minutes at 30°C to induce the expression of Ubr1. Cells were shifted to 37°C in YEP galactose containing nocodazole and doxycycline to deplete Pds5 and/or Scc3. (A) Chromosomal spreads were prepared at the indicated time points and stained with DAPI for DNA, anti-HA (mouse, 16B12) and anti-MYC (rabbit, 71D10) antibodies. The secondary antibodies were Alexa Fluor 488 anti-mouse and Alexa Fluor 568 anti-rabbit. Protein fluorescence was quantified using Metamorph software. At every time point fluorescence of 50 nuclei was determined. Error bars represent standard deviation. (B) Western blot of TCA protein extracts probed with anti-HA (16B12), anti-MYC (71D10) and anti-Cdc28 (sc-28550, Santa Cruz). (C) FACS analysis of cellular DNA content. (D–F) Strains were staged in G1 with <i>α</i>-factor in YEP raffinose at 30°C, resuspended in YEP galactose containing <i>α</i>-factor and incubated for 45 minutes at 30°C to induce the expression of Ubr1. Cells were then shifted to 37°C in YEP galactose containing doxycycline and <i>α</i>-factor, incubated for 90 minutes to deplete Pds5 and/or Scc3 and subsequently released in YEP galactose containing nocodazole and doxycycline at 37°C. Chromosomal spreads (D), Western blot (E), and FACS analysis of cellular DNA content (F) are shown.</p

Cohesin rings devoid of Scc3 and Pds5 topologically embrace circular DNA.

<p>Strains 1021 (untagged), 1813 (<i>SCC3-HA6</i>, <i>SCC1-Myc18</i>), 1625 (<i>SCC3-HA6-</i>degron, <i>SCC1-Myc18</i>), 2525 (<i>PDS5-HA6</i>, <i>SCC1-Myc18</i>) and 1818 (<i>PDS5-HA6</i>-degron, <i>SCC1-Myc18</i>) carried the centromeric minichromosomes. (A) Yeast lysates were incubated with BglII restriction enzyme as indicated. Minichromosomes were co-immunoprecipitated with Scc1-Myc18. DNA was prepared by phenol/chloroform extraction and separated on a 1% agarose gel with ethidium bromide. Southern blot probed with a <i>TRP1</i>-specific probe is shown. Nicked (N), linear (L), and closed circular (C) forms of the minichromosome are indicated. (B) Minichromosomes were immunoprecipitated with anti-HA antibody. Minichromosomes from <i>SCC3-HA6</i> but not <i>SCC3-HA6</i>-degron strains could be co-immunoprecipitated with Scc3 indicating the efficient depletion of Scc3 from the minichromosomes in the <i>SCC3-HA6</i>-degron strain. Since Pds5 association with minichromosomes is very salt-sensitive, they could not be co-immunoprecipitated with Pds5-HA6 in either the wild type or <i>PDS5-HA6</i>-degron strains under our experimental conditions.</p

Interaction of Pds5, Scc3 and Wpl1 with cohesin ring.

<p>Lysates of nocodazole/benomyl arrested yeast cultures were incubated with IgG sepharose to precipitate Scc1-TAP or Smc3-TAP. The presence of different proteins on the IgG beads was analysed by Western blot probed with anti-HA (12CA5), anti-MYC (71D10) and PAP (P1291, Sigma). The strains were in (A): 1771 (<i>SCC3-MYC18, PDS5-HA6</i>), 1829 (<i>SCC3-MYC18, PDS5-HA6</i>-degron, <i>SCC1-TAP</i>), 1958 (<i>SCC3-MYC18, PDS5-HA6, SCC1-TAP</i>); in (B): 1734 (<i>PDS5-MYC18, SCC3-HA6</i>), 1834 (<i>PDS5-MYC18, SCC3-HA6</i>-degron, <i>SCC1-TAP</i>), 1956 (<i>PDS5-MYC18, SCC3-HA6, SCC1-TAP</i>); in (C): 1882 (<i>WPL1-MYC18, PDS5-HA6</i>), 2014 (<i>WPL1-MYC18, PDS5-HA6, SCC1-TAP</i>), 2016 (<i>WPL1-MYC18, PDS5-HA6</i>-degron, <i>SCC1-TAP</i>); in (D): 1880 (<i>WPL1-MYC18, SCC3-HA6</i>), 2012 (<i>WPL1-MYC18, SCC3-HA6, SCC1-TAP</i>), 2018 (<i>WPL1-MYC18, SCC3-HA6</i>-degron, <i>SCC1-TAP</i>); in (E): 1771 (<i>SCC3-MYC18, PDS5-HA6</i>), 2251 (<i>SCC3-MYC18, PDS5-HA6, SMC3-TAP</i>), 2290 (<i>SCC3-MYC18, PDS5-HA6</i>-degron, <i>SMC3-TAP</i>); in (F): 1734 (<i>PDS5-MYC18, SCC3-HA6</i>), 2249 (<i>PDS5-MYC18, SCC3-HA6, SMC3-TAP</i>), 2264 (<i>PDS5-MYC18, SCC3-HA6</i>-degron, <i>SMC3-TAP</i>); in (G): 1882 (<i>WPL1-MYC18, PDS5-HA6</i>), 2253 (<i>WPL1-MYC18, PDS5-HA6, SMC3-TAP</i>), 2265 (<i>WPL1-MYC18, PDS5-HA6</i>-degron, <i>SMC3-TAP</i>); in (H): 1882 (<i>WPL1-MYC18, PDS5-HA6</i>), 2261 (<i>WPL1-MYC18, SCC3-HA6, SMC3-TAP</i>), 2271 (<i>WPL1-MYC18, SCC3-HA6</i>-degron, <i>SMC3-TAP</i>).</p

A model of how Scc3 and Pds5 play a role in the establishment of sister chromatid cohesion but are not required to stabilize cohesin rings on the DNA.

<p>In the normal cell cycle of budding yeast Scc1 subunit is synthesized in the late G1 or early S phase. Scc1 binds to Smc1/Smc3 heterodimer and completes the cohesin ring. Scc3 and Pds5 stably associate with cohesin via Scc1 subunit. Cohesin complexes are loaded on the chromosomes. During DNA replication two newly generated sister chromatids are captured inside a single cohesin ring in a process which remains poorly understood. Scc3 and Pds5 function to ensure that two sister chromatids are captured inside a cohesin ring. In their absence, cohesin complexes are stably loaded on the DNA but fail to embrace both of the sister chromatids resulting in defective sister chromatid cohesion (A). We can speculate that Pds5 and Scc3 could stabilize cohesin rings specifically during the replication fork passage (B) or transiently bind sister chromatids during the establishment of cohesion (C). Alternatively they could mediate a transient interaction between two cohesin rings as proposed by <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002856#pgen.1002856-Zhang2" target="_blank">[61]</a> (D).</p