Identification of ORC1/CDC6-interacting factors in Trypanosoma brucei reveals critical features of origin recognition complex architecture

Abstract

DNA replication initiates by formation of a pre-replication complex on sequences termed origins. In eukaryotes, the pre-replication complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1-6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing.

Figure 1. MCM helicase subunits in T. brucei and co-expression with ORC1/CDC6 as epitope tagged variants.

A. An unrooted phylogenetic tree is shown, detailing the homology between predicted MCM helicase subunits in T. brucei (Tbr) relative to orthologues in H. sapiens (Hsa), S. cerevisiae (Sce) and A. thaliana (Ath). Complete protein sequences were aligned with ClustalX, using default settings, and the phylogenetic tree was displayed using TreeView (Page, 1996); the distance corresponding to 10 amino acid changes per 100 positions is indicated (0.1) B. A diagrammatic representation of the MCM helicase subunits in T. brucei. The length of the predicted polypeptides is shown (in amino acid residues), and the position of conserved functional motifs are indicated: an N-terminal Zinc Finger (Zn, blue box); and Walker A and B boxes (A and B, red boxes), an Arginine finger (R, orange box) and sensor 1 and 2 motifs (S1 and S2, green boxes), all involved in nucleotide binding and hydrolysis. C. Western blots of procyclic form TREU 927 T. brucei cells co-expressing C-terminally HA-tagged TbMCM subunits (MCM-HA) and C-terminally Myc-tagged TbORC1/CDC6. The upper panel shows TbMCM-HA expression in whole cell extracts, detected using anti-HA antibody, and the bottom panel shows TbORC1/CDC6-Myc from the same whole cell extracts detected using anti-Myc antibody. Single clones are shown for TbMCM4-HA and TbMCM6-HA, two clones for TbMCM2-HA and TbMCM7-HA, and three clones for TbMCM3-HA. Size markers (kDa) are indicated.

Figure 2. Mass spectrometric characterisation of T. brucei TbMCM-HA immunoprecipitates and yeast two hybrid analysis reveals MCM subunit interactions.

A. Eluates are shown from immunoprecipitations (IP) using anti-HA antibody from T. brucei cell lines co-expressing TbORC1/CDC6-Myc and TbMCM3-HA, TbMCM-6HA or TbMCM7-HA; as a control an IP eluate from a cell expressing TbORC1/CDC6-Myc, but no HA-tagged protein, is also shown. Proteins in the IP eluates were separated by SDS-PAGE and visualised by colloidal commassie staining; size markers are shown and Ig indicates immunoglobulin polypeptides. Bands that were excised and analysed by mass spectrometry are numbered; the results of this analysis are shown in B, where the number of unique peptides identified for each band is shown, as well as the T. brucei gene ID and MCM subunit. C. Inter-MCM subunit interactions were examined by yeast 2 hybrid analysis. Growth of yeast clones co-expressing individual TbMCM subunits (numbered 2–7, indicating TbMCM2–7) as fusions with the Gal DNA binding domain (pGBK-MCM) and with the Gal activating domain (pGAD-MCM) is shown; as a control, growth of the fusion protein-expressing plasmids are shown when co-transformed with pGBK or pGAD vectors without any MCM gene insert (V). Growth on minimal medium lacking tryptophan, leucine and histidine (-T-L-H) indicates weak interaction, while growth on the same media supplemented with Aureobasidin A (-T-L-H+AbA) or 3′ aminotriazole (-T-L-L+3-AT), indicates strong interaction; growth on medium lacking only tryptophan and lecuine (-T-L) shows that the cells that cannot grow through interaction are viable. D shows a model for the assembly and subunit architecture of the MCM hexamer in eukaryotes; a putative subunit complex identified by IP in this analysis is indicated (dashed box), while intersubunit interactions revealed by yeast 2 hybrid analysis are shown in the putative heterohexamer (single- and doubled-headed arrows denote one- and bi-directional interactions, respectively, and strong and weak interactions are distinguished by solid and dashed lines, respectively).

Figure 3. Western blot analysis of TbORC1/CDC6-Myc and TbMCM-HA immunoprecipitations.

A. 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 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.

Figure 4. Identification of a T. brucei ORC1/CDC6-interacting protein as a putative orthologue of eukaryotic Orc4.

A. 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 A sequence comparison of the predicted Tb13380 polypeptide (translation of T. brucei 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: H. sapiens (Hsa, O43929), D. melanogaster (Dme, AAF47276.1), A. thaliana (Ath, CAE01428), S. cerevisiae (Sce, P54791), and T. thermophila (Tth, 51.m00235). Also shown are putative Orc4 orthologues from further species: P. falciparum (Pfa, PF13_0189), Dictyostelium discoideum (Ddi, DDB0168430), Cryptosporidium parvum (Cpa, cgd2_1550), Theileria annulata (Tan, TA12985), Giardia lamblia (Gla, ctg02_3) and Encephalitozoon cuniculi (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).

Figure 5. Co-immunoprecipitation demonstrates interaction between TbORC1/CDC6 and two novel factors.

Input (I) and eluate (E) samples from immunoprecipitations (IPs) using antibody against HA (anti-HA) are shown from procyclic T. brucei whole cell extracts of cells co-expressing TbORC1/CDC6-Myc (ORC-myc) and either Tb7980-HA (IP labelled ORC1/CDC6-7980) or Tb3120-HA (IP labelled ORC1/CDC6-3120); a control anti-HA IP is shown from cells expressing only Myc-tagged TbORC1/CDC6. Samples were separated by SDS-PAGE, transferred to a nylon membrane and probed with anti-HA antibody (upper panel) or with anti-Myc antibody (lower panel). Bands corresponding with immunoglobulin heavy chain (Ig), an anti-HA cross-reacting band (*) and with either HA-tagged Tb7980 or Tb3120 are indicated in the HA IP samples; size markers (kDa) are indicated.

Figure 6. Effect of TbORC1/CDC6, TbORC4, Tb7980 and Tb3120 RNAi on procyclic form T. brucei.

A. Analysis of nuclear (N) and kinetoplast (K) DNA configurations in procyclic form T. brucei cells 4 and 6 days post-RNAi induction (induced by tetracycline; Tet+) against TbORC1/CDC6, TbORC4 (13380), Tb7980 and Tb3120; for each gene, N and K configurations are also 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 (others). In each graph, the insert shows the extent of loss of cognate mRNA by quantitative reverse transcriptase PCR (qRT-PCR): levels of mRNA are shown four days after RNAi induction (Tet+) relative to the uninduced cells (Tet−), where qRT-PCR amplification has been set as 1.0 (values are the mean of four experimental repetitions and vertical lines denote standard deviation). B Representative images, showing aberrant cells with 0N1K DNA configuration seen 6 days after induction of RNAi of the gene indicated. Cells are shown with DNA stained by DAPI (N and K are arrowed), and as merge of DAPI and phase contrast images.

Figure 7. RNAi of TbORC1/CDC6, TbORC4 and Tb7980 in bloodstream form T. brucei cells results in rapid growth arrest.

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

Figure 8. Effect of TbORC1/CDC6, TbORC4 and Tb7980 RNAi on bloodstream form T. brucei.

A. Analysis of nuclear (N) and kinetoplast (K) DNA configurations in bloodstream form T. brucei cells at time points following RNAi induction (induced by tetracycline; Tet+) against TbORC1/CDC6, TbORC4 (13380) and Tb7980; 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. 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).

Figure 9. Origin Recognition Complex architecture in the eukaryotes S. cerevisiae, T. brucei and N. gruberi.

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 S. cerevisiae based on work by Chen et al ; the specific arrangement of Orcs 2–5 is inferred from Moreno del-Alamo . In T. brucei, recognisable ORC subunit orthologues are identified, while subunits that are absent or highly diverged are shown as dotted circles containing question marks. The T. brucei 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 N. gruberi, a free-living relative of T. brucei, are shown for comparison; here again, Orc1 appears to be an Orc1-Cdc6 fusion.