Multiple genetic networks in recombination-mediated <i>VSG</i> variation.

<p>The diagram shows the active ES (ES1) expressing <i>VSG</i> (red) and a silent ES containing <i>vsg</i> (green). RMI1 deficiency exhibited opposite phenotype in <i>VSG</i>-GC and ES GC switching, suggesting that molecular mechanisms of these events are distinct. We propose that there may be ‘replication fork instability zone(s)’ near <i>VSG</i>, potentially the 70-bp repeats, but not in ES promoter region. The RMI1-TOPO3α is required to dissolve recombination intermediates arising in this zone during replication. When their function is compromised, recombination intermediates accumulate and result in <i>VSG</i>-GC and crossover switches (a). RMI1 is required to promote ES GC. As ES GC requires extension of much longer regions compared to <i>VSG</i> GC, having a stable replication fork must be advantageous than having a migrating D-loop. Budding yeast SGS1 is required for replisome stability. Therefore, we propose that <i>T. brucei</i> RMI1-TOPO3α might stabilize a newly assembled replication fork between the active and a silent ES near promoter region. Formation of a stable replication fork may be the key step to generate ES GC switching (b).</p

TbRMI1 monitors <i>VSG</i> recombination.

<p>TbRMI1 deficiency increases <i>VSG</i> GC and crossover (XO) but decreases ES GC. 275 cloned switchers were examined and the frequency of each switching mechanism was plotted. White bars are wild type and dark grey bars are <i>rmi1</i> mutant. Error bars indicate standard deviation. The results are also summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025313#pone-0025313-t001" target="_blank">Table 1</a>. Switched variants were assigned as follows (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025313#pone-0025313-g003" target="_blank">Figure 3A</a> diagram); <i>VSG</i>-GC switchers, <i>BSD⁺ VSG</i> 427-2<i>^-</i>; ‘ES GC or ES loss’ switchers, <i>BSD^- VSG</i> 427-2<i>^-</i>; crossover switchers, <i>BSD⁺ VSG</i> 427-2<i>⁺</i>; <i>in situ</i>, <i>BSD⁺ VSG</i> 427-2<i>⁺</i>.</p

TbRMI1 deficiency increases <i>VSG</i> switching frequency.

<p>(A) <i>VSG</i> switching reporter cell line and switching mechanisms. The active expression site, ES1-<i>VSG</i> 427-2, was doubly marked with <i>BSD</i> at the promoter and <i>PUR-TK</i> immediately at the 3′ end of the 70-bp repeats. <i>VSG</i> switching events will accompany either loss or repression of the <i>PUR-TK</i> marker, which allows only the switchers to grow in the presence of GCV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025313#pone.0025313-Kim1" target="_blank">[39]</a>. Non-switchers can be eliminated as described in the text. Black circles are telomere repeats. (B) TbRMI1 deficiency increases <i>VSG</i> switching frequency. Switching frequency was measured in wild type, <i>rmi1</i>, and <i>rmi1</i> cells transfected either with an empty vector or the wild-type <i>RMI1</i>.</p

Strains used in this study.

<p>(*) The full annotation is Tb927.8.2040.</p

Tb927.3.1830 interacts with TbTOPO3α.

<p>(A) Tb927.3.1830-HA co-immunoprecipitates with TOPO3α-MYC. (B) TOPO3α-MYC co-immunoprecipitates with Tb927.3.1830-HA. TOPO3α was endogenously tagged with 3xMYC, and Tb927.8.2040 and Tb927.3.1830 with 3xHA. Cell lysates were immunoprecipitated either with anti-HA or anti-MYC antibodies and analyzed by western blot. (*) and (**) indicate antibody heavy and light chains. Arrow heads indicate break-down products of 1830-HA.</p

TbRMI1 is required for both RAD51-dependent and RAD51-independent <i>VSG</i> switching.

<p>(A) Simultaneous deletion of <i>RMI1</i> and <i>RAD51</i> severely impairs <i>VSG</i> switching. Overall <i>VSG</i> switching frequencies of wild type, <i>rmi1</i>, <i>rad51</i>, and <i>rmi1 rad51</i> were plotted. (B) Increased <i>VSG</i> GC rate in <i>rmi1</i> was dependent on RAD51, but RMI1 also functions in <i>VSG</i> GC switching independently of RAD51. (C) Increased crossover in <i>rmi1</i> was dependent on RAD51. (D) ‘ES GC or ES loss’ switching was impaired in a <i>rad51</i> or <i>rmi1</i> single mutant. The results are also summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025313#pone-0025313-t001" target="_blank">Table 1</a>.</p

Genetic interaction between <i>T. brucei RMI1</i> and <i>TOPO3α</i>.

<p>(A) <i>TbRMI1</i> is epistatic to <i>TbTOPO3α</i> in <i>VSG</i> switching. (B) <i>RMI1</i> absence increases gene-conversion frequency at <i>URA3</i> locus and <i>TbRMI1</i> is epistatic to <i>TbTOPO3α</i> in gene conversion at this locus. Two counter-selectable markers, <i>TK</i> and <i>URA3</i>, were used to measure the GC rates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025313#pone.0025313-Kim1" target="_blank">[39]</a>. <i>GCV^R</i> and <i>FOA^R</i> clones were counted and fold increase relative to wild type was plotted. Error bars indicate standard deviation. (*) in Figure 5B indicates <i>topo3α</i> data taken from the previous report <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025313#pone.0025313-Kim1" target="_blank">[39]</a>. Unpaired T tests were done and means of switching and GC frequencies were significantly different in the mutants.</p

Deletion of Tb927.3.1830 causes a growth defect.

<p>(A) Alignment of Tb927.3.1830 with human, mouse and chicken RMI1. DUF1767 and OB-fold domains are indicated in pink and yellow boxes, respectively. Conserved lysine residues are in green box in the OB1 domain. Blue bars show locations of five β-strands of OB1. (B) Deletion scheme for Tb927.3.1830. The entire open reading frames (ORFs) of Tb927.3.1830 were sequentially deleted using cassettes containing <i>HYG-TK</i> or <i>PUR</i> flanked by loxP sites. The markers were removed by transient expression of Cre-recombinase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025313#pone.0025313-Kim1" target="_blank">[39]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025313#pone.0025313-Scahill1" target="_blank">[64]</a>. (C) <i>Tbrmi1</i> exhibits a minor growth defect, similar to <i>Tbtopo3α</i>. Wild-type, <i>rmi1^-/+</i> and <i>rmi1^-/-</i> cells were diluted to 10,000 cells/ml and cells were counted after two days of incubation. This was repeated twice. Growth phenotypes of <i>Tbtopo3α</i> mutants were taken from the previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025313#pone.0025313-Kim1" target="_blank">[39]</a>. Error bars are shown, but are small. (D) Diagrams of human RMI1/2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025313#pone.0025313-Hoadley1" target="_blank">[41]</a> and Tb927.3.1830.</p

LOS1 shows sequence similarities to the MCM-Binding Protein (MCM-BP).

<p>(A) Alignment of LOS1 with human, fish, worm and plant MCM-BP is shown. BLAST searches identify that all of MCM-BP including LOS1 contain two families of sequences, called ‘MCM Bind Superfamily’ (underlined in blue) and ‘Racemase 4 Super family’ (underlined in red). (B) A schematic diagram of <i>T. brucei</i> MCM-BP. A black triangle in the ‘Racemase 4 Superfamily’ domain indicates the site where a <i>mariner</i> transposon (Tn) was inserted.</p

TbMCM-BP co-purifies with MCM4-MCM8.

<p>(A) TbMCM-BP interacts with MCM5 but not with MCM2 by co-immunoprecipitation. (B) Allele modification in cells exclusively expressing TbMCM-BP tagged with PTP. (C) Immunoblot monitoring of TbMCM-BP-PTP tandem affinity purification in extract (Input), IgG affinity chromatography flowthrough (FT-IgG), TEV protease eluate (Elu TEV), flowthrough of the anti-ProtC affinity chromatography (FT-ProtC), and final eluate (Elu). Note that TEV protease-mediated removal of the ProtA domains led to a size reduction of tagged TbMCM-BP (‘BP-PTP’ to ‘BP-P’). Relative amounts of aliquots are specified by x values. (D) Tandem affinity purification of TbMCM-BP. Final eluate and smaller aliquots of the TEV protease eluate and extract were separated on an SDS–gradient polyacrylamide gel and stained with Coomassie blue or Sypro Ruby. (E) TbMCM-BP is localized in the nucleus throughout the cell cycle in PF.</p