BRCA2 variants and polypeptide fusions to assess the function of the BRC repeats in recombination and VSG switching

A. Functional domains of BRCA2 variants and polypeptide fusions analysed by expression in bloodstream stage −/− mutants are shown. Full-length BRCA2 may contain up to 15 BRC repeats, a conserved DSS1-DNA-binding domain (DBD) and two putative nuclear localization signals (NLSs). BRCA2 displays conservation of the DBD, but has only one BRC repeat and NLSs have not been predicted. 1BRC differs from full-length BRCA2 only in reduction of the BRC array to a single repeat. BRCrep is a polypeptide fragment of BRCA2 encompassing the BRC repeats and 33 downstream amino acids, including a bipartite NLS. BRC–RPA is a fusion of the BRCrep polypeptide to the 50 kDa replication protein A subunit. B. Homologous recombination efficiency was assayed by determining the number of transformants recovered (per 10 cells put on antibiotic selection) when the construct tub--tub was electroporated into wild-type (WT) cells, −/− mutants and −/− cells expressing the BRCA2 variant polypeptides detailed above (−/−/+). C. VSG-switching frequencies of WT (Lister 427) cells, −/− mutants and −/− cells expressing the BRCA2 variant polypeptides are shown. Values are the means of at least three independent experiments, and bars represent standard error.<p><b>Copyright information:</b></p><p>Taken from " BRCA2 acts in antigenic variation and has undergone a recent expansion in BRC repeat number that is important during homologous recombination"</p><p></p><p>Molecular Microbiology 2008;68(5):1237-1251.</p><p>Published online Jan 2008</p><p>PMCID:PMC2408642.</p><p>© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd</p

Functions of Dot1-Mediated Histone H3 Methylation

<p>Two models for the role of Dot1-mediated methylation of histone H3 are diagrammed, comparing a Dot1 mutant (ΔDot1) and a wild-type cell. The repulsion model is derived from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0060185#pbio-0060185-b021" target="_blank">21</a>]. Methylation of histone H3 is indicated by “me”, and the level of transcription of a chromosome (black line) is indicated by a shaded gray bar (a thick bar indicates active transcription, a thin bar indicates silenced transcription). Silencing factors (such as Sir proteins in yeast; light blue circles) are indicated localised to the telomere (vertical line) in wild-type cells, being excluded from elsewhere by H3K79 methylation. Mutation of Dot1 removes H3K79 methylation, de-repressing transcription of the telomeric region. The recruitment model is based on [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0060185#pbio-0060185-b032" target="_blank">32</a>], and shows the same region of chromosome after suffering a DNA double-strand break (gap in the line). Here, histone H3K79 methylation recruits a checkpoint signalling factor (Rad9 in yeast; dark blue circle), and in the absence of histone H3K79 methylation processing of the DNA break to yield single stranded DNA is increased, amplifying the DNA damage signalling cascade.</p

Mechanisms of <i>VSG</i> Switching during Antigenic Variation in T. brucei

<p>The <i>VSG</i> gene expressed prior to a switch (indicated by a blue box) is transcribed from an expression site (ES) that is found at the telomere (vertical black line) of a chromosome (horizontal black line); active transcription of the ES is indicated by a dotted arrow, <i>ESAG</i>s are depicted by black boxes, and 70-bp repeat sequence is shown as a hatched box. Gene conversion to generate a <i>VSG</i> switch can occur by copying a silent <i>VSG</i> (red box) from a subtelomeric array into the ES, replacing the resident <i>VSG</i>; the amount of sequence copied during gene conversion is illustrated, and normally encompasses the <i>VSG</i> ORF and extends upstream to the 70-bp repeats. The silent <i>VSG</i> donor can also be telomeric (either in a mini chromosome or in an inactive ES); here, the downstream limit of conversion can extend to the telomere repeats, while the upstream limit can either be in the 70-bp repeats or the <i>ESAG</i>s (if the donor is in an ES). Segmental <i>VSG</i> conversion involves the copying of sequence from multiple, normally nonfunctional <i>VSG</i>s (pink, red, or green boxes) to generate a novel mosaic <i>VSG</i> in the ES. In transcriptional <i>VSG</i> switching, recombination appears not to be involved; instead, limited transcription at a silent <i>VSG</i> ES (indicated by a small arrow) becomes activated to generate fully active transcription, while the previously active ES is silenced.</p

<i>VSG</i> Switching Hierarchy in T. brucei

<p>The graph is adapted from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0060185#pbio-0060185-b009" target="_blank">9</a>] and shows the numbers of T. brucei cells (parasitaemia) measured in a cow for up to 70 days post-infection (this measurement is depicted by inversely plotting the prepatent period, in days, that a 0.2-ml inoculum of cattle blood achieves a parasitaemia of 1 × 10^8.1 trypanosomes ml⁻¹ units in an immunosuppressed mouse). Below the graph is a depiction of <i>VSG</i> gene activation timing (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0060185#pbio-0060185-g002" target="_blank">Figure 2</a> for details of the switch mechanisms). During <i>VSG</i> switches driven by recombination, silent <i>VSG</i>s at a telomere are, in general, activated more frequently that subtelomeric array <i>VSG</i>s, which are activated more frequently than <i>VSG</i> pseudogenes (pseudo). It is unclear (indicated by a question mark) if transcriptional switches between <i>VSG</i> bloodstream expression sites (BES) occur predominantly at the start of an infection or continue throughout.</p

Additional file 1: of Genome-wide mapping reveals single-origin chromosome replication in Leishmania, a eukaryotic microbe

Supplementary methods and supporting information (11 figures and 1 table, each of which is referred to and explained in the main paper). (PDF 68032 kb

Origin Recognition Complex architecture in the eukaryotes <i>S. cerevisiae</i>, <i>T. brucei</i> and <i>N. gruberi</i>.

<p>The architecture of the Origin Recognition Complex (ORC; composed of Orc subunits numbered 1–6), bound to the Orc1-related factor Cdc6 and to DNA (black line), is shown for <i>S. cerevisiae</i> based on work by Chen et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032674#pone.0032674-Chen1" target="_blank">[22]</a>; the specific arrangement of Orcs 2–5 is inferred from Moreno del-Alamo <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032674#pone.0032674-MorenodelAlamo1" target="_blank">[58]</a>. In <i>T. brucei</i>, recognisable ORC subunit orthologues are identified, while subunits that are absent or highly diverged are shown as dotted circles containing question marks. The <i>T. brucei</i> ORC subunit indicated as Orc1 appears to be a bi-functional Orc1-Cdc6 protein, and it is unknown if it therefore occupies a distinct architectural position in the ORC or adopts a distinct structure. Putative ORC subunits identified bioinformatically in <i>N. gruberi</i>, a free-living relative of <i>T. brucei</i>, are shown for comparison; here again, Orc1 appears to be an Orc1-Cdc6 fusion.</p

Western blot analysis of TbORC1/CDC6-Myc and TbMCM-HA immunoprecipitations.

<p><b>A.</b> Input (I) and eluate (E) samples from immunoprecipitations (IPs) from procyclic form whole cell extracts using antibody against HA are shown from cells co-expressing TbORC1/CDC6-Myc (ORC-myc) and TbMCM3-HA, TbMCM6-HA or TbMCM7-HA, as well as from control cells expressing only Myc-tagged TbORC1/CDC6. Samples were separated on a 10% SDS-PAGE gel, transferred to a membrane and probed with anti-HA antibody (upper panel) or with anti-Myc antibody (lower panel). <b>B</b> shows the reciprocal experiment in which IP was performed with anti-Myc antibody from cells co-expressing TbORC1/CDC6-Myc and TbMCM6-HA or TbMCM7-HA, and from control cells expressing only HA-tagged MCM6 or MCM7. Size markers (kDa) are indicated.</p

Effect of TbORC1/CDC6, TbORC4 and Tb7980 RNAi on bloodstream form <i>T. brucei</i>.

<p><b>A.</b> Analysis of nuclear (N) and kinetoplast (K) DNA configurations in bloodstream form <i>T. brucei</i> cells at time points following RNAi induction (induced by tetracycline; Tet+) against <i>TbORC1/CDC6</i>, <i>TbORC4 (13380)</i> and <i>Tb7980</i>; for comparison, N and K configurations are shown in cells without RNAi induction (Tet−). Graphs depict the proportion of cells (derived by counting >200 DAPI-stained cells) with conventional 1N1K, 1N2K, or 2N2K configurations, or with any aberrant configuration (grouped as others). <b>B.</b> FACS profiles of propidium iodide (PI)-stained cells after RNAi induction (Tet+) are shown as histograms after FACS sorting, sampled at the time points post-induction (control cells, without RNAi induction (Tet−), are shown sampled at the time shown, corresponding to growth from an equivalent starting density to the RNAi- induced cells). Peaks corresponding with cells containing 2C and 4C DNA content are indicated, as is the peak position for cells with 8C content (C represents haploid DNA content).</p

RNAi of TbORC1/CDC6, TbORC4 and Tb7980 in bloodstream form <i>T. brucei</i> cells results in rapid growth arrest.

<p>Growth curves are shown for bloodstream form <i>T. brucei</i> cells in the absence or presence of tetracycline (tet−, shown as solid line, and tet+, dashed line, respectively), which induces RNAi, targeting either <i>TbORC1/CDC6</i>, <i>TbORC4 (Tb13380)</i>, or <i>Tb7980</i> mRNA. For each factor, cell density over time was examined in two clonal RNAi cell lines (identified by C).</p

Identification of a <i>T. brucei</i> ORC1/CDC6-interacting protein as a putative orthologue of eukaryotic Orc4.

<p><b>A.</b> Input (I) and eluate (E) samples from immunoprecipitations (IPs) from procyclic form whole cell extracts are shown using antibody against HA (anti-HA) or against Myc (anti-Myc). Anti-HA IP was performed from cells co-expressing TbORC1/CDC6-Myc (ORC-myc) and Tb13380-HA, or from control cells expressing only Myc-tagged TbORC1/CDC6; anti-Myc IP was from cells co-expressing TbORC1/CDC6-Myc and Tb13380-HA, or from control cells expressing only HA-tagged Tb13380. In all cases IP samples were separated on a 10% SDS-PAGE gel, transferred to a nylon membrane and probed with anti-HA antibody (upper panel) or with anti-Myc antibody (lower panel). Size markers (kDa) are indicated. <b>B</b> A sequence comparison of the predicted Tb13380 polypeptide (translation of <i>T. brucei</i> gene ID Tb927.10.13380) with Orc4 proteins from a number of eukaryotes (black and grey boxing highlights residues identical or conserved, respectively, in 50% of the sequences). For the following species Orc4 has been functionally or bioinformatically identified: <i>H. sapiens</i> (Hsa, O43929), <i>D. melanogaster</i> (Dme, AAF47276.1), <i>A. thaliana</i> (Ath, CAE01428), <i>S. cerevisiae</i> (Sce, P54791), and <i>T. thermophila</i> (Tth, 51.m00235). Also shown are putative Orc4 orthologues from further species: <i>P. falciparum</i> (Pfa, PF13_0189), <i>Dictyostelium discoideum</i> (Ddi, DDB0168430), <i>Cryptosporidium parvum</i> (Cpa, cgd2_1550), <i>Theileria annulata</i> (Tan, TA12985), <i>Giardia lamblia</i> (Gla, ctg02_3) and <i>Encephalitozoon cuniculi</i> (Ecu, NP_59761). The Tb13380 (ORC4) polypeptide is shown diagrammatically (number of amino acid residues is indicated), highlighting regions of conservation around motifs involved in nucleotide binding and hydrolysis: Walker A and B boxes (A and B, red boxes), an Arginine finger (R, orange box) and a Sensor 1 motif (S1, green boxes).</p