DOI: 10.5061/dryad.wh70rxwnv
title: Genomic analysis finds no evidence of canonical eukaryotic DNA processing complexes in a free-living protist

description: Cells replicate and segregate their DNA with precision. Previous studies
 showed that these regulated cell-cycle processes were present in the last
 eukaryotic common ancestor and that their core molecular parts are
 conserved across eukaryotes. However, some metamonad parasites have
 secondarily lost components of the DNA processing and segregation
 apparatuses. To clarify the evolutionary history of these systems in these
 unusual eukaryotes, we generated a genome assembly for the free-living
 metamonad Carpediemonas membranifera and carried out a comparative
 genomics analysis. Here, we show that parasitic and free-living metamonads
 harbor an incomplete set of proteins for processing and segregating DNA.
 Unexpectedly, Carpediemonas species are further streamlined, lacking the
 origin recognition complex, Cdc6 and most structural kinetochore subunits.
 Carpediemonas species are thus the first known eukaryotes that appear to
 lack this suite of conserved complexes, suggesting that they likely rely
 on yet-to-be-discovered or alternative mechanisms to carry out these
 fundamental processes.
description: The data here is fully described in the source manuscript.
description: Supplementary sequences: Sequences
 corresponding to ‘Supplementary Data 4. Spindle assembly, kinetochore and
 APC/C orthologs in 18 diverse eukaryotic genomes’. Each multifasta
 sequence file is labeled according to respective aliases reported in the
 table. 'Orc1_Cdc6_Orc1-Cdc6-like.fasta' file
 is a multifasta sequence file corresponding to ‘Supplementary Data 6.
 Orc1, Cdc6 and Orc1/Cdc6-likeproteins. Information used in Supplementary
 Figure 3, panels b and d’ The figures presented here
 correspond to high resolution versions of the figures presented in the
 Supplementary Information of the source manuscript.
 Supplementary figure legends: Supplementary
 Fig. 1: Maximum-likelihood reconstruction of the phylogenetic
 relationships within the Metamonada clade. An initial reconstruction was
 carried out in IQ-Tree with the LG+C60+F+Γ model and 1000 ultrafast
 bootstraps, this was followed by tree inference under LG+PMSF(C60)+F+ Γ
 model using 100 nonparametric bootstraps; alignment length of 181 genes
 encompassing 48341 sites. Tree rooted on the ancestral branch of Amorphea.
 Scale bar shows the inferred number of amino acid substitutions per site.
 Bootstrap values are represented as shaded dots on each branch, and the
 values are represented in the following order: SH-aLRT support
 percentage/aBayes/nonparametric bootstrapping. Supplementary
 Fig. 2: Phylogenetic reconstruction of Orc5 proteins inferred with
 IQ-TREE<sup>15</sup> under the LG+ C60+F+ Γ model using 1000
 ultrafast bootstraps (SH-aLRT support percentage/aBayes/bootstrap). Value
 ranges for branches are shown by dots, the red dot indicates that the
 values apply for each node within the clade. The alignment consists of 60
 taxa with 422 sites after trimming. For simplicity, only the domain
 architecture for metamonads, <i>S. cerevisiae</i>,<i> A.
 thaliana</i> and <i>H. sapiens</i> are depicted on the
 tree. Supplementary Fig. 3: Orc1-6 and
 Cdc6 proteins. (a) Left: typical domain architecture observed for Orc1-6
 and Cdc6 in <i>Saccharomyces cerevisiae</i>. Right:
 representative domain architecture of metamonad proteins drawn to reflect
 the most common protein size. If no species name is given, then the
 depicted domain structure was found in all of the metamonads where
 present. Numbers on the right of each depiction correspond to the total
 protein length or its range in the case of metamonads (additional
 information in Supplementary Data 2). (b) Comparison of Orc1, Cdc6 and
 Orc1/Cdc6-like protein lengths across 81 eukaryotes encompassing
 metamonads and non-metamonads protists (source information in
 Supplementary Data 6). Metamonad proteins are highlighted with green
 shaded bubbles in the background. (c) Orc1/Cdc6 partial ATPase domain
 showing Walker A and Walker B motifs including R-finger. Reference species
 at the top. Multiple sequence alignment was visualized with
 Jalview<sup>72</sup> using the Clustal colouring scheme. (d)
 Phylogenetic reconstruction of Orc1, Cdc6 and Orc1/Cdc6-like proteins
 inferred with IQ-TREE<sup>15</sup> under the LG+ C10+F+ Γ
 model using 1000 ultrafast bootstraps (bootstrap value ranges for branches
 are shown with black and grey dots). The alignment consists of 81 taxa
 with 367 sites after trimming. Orc1/Cdc6-like proteins do not form a clade
 with <i>bona fide</i> Orc1 and Cdc6 proteins making it
 impossible to definitively establish whether or not they are
 orthologs. Supplementary
 Fig. 4: The distribution of core molecular systems of the replisome,
 double strand break repair and endonucleases in nucleomorph genomes of
 cryptophyte and chlorarachniophytes. Supplementary
 Fig. 5: The distribution of core molecular systems of DNA repair across
 eukaryotic diversity. A schematic global eukaryote phylogeny is shown on
 the left with classification of the major metamonad lineages indicated.
 Double strand break repair and endonuclease sets. ***Carpediemonas-Like
 Organisms. ‘?’ is used in cases where correct orthology was difficult to
 establish, so the protein name appears with the suffix ‘-like’ in
 tables.  <br clear="all"> Supplementary Fig. 6: Presence/absence diagram
 of LECA kinetochore components in eukaryotes, with a greater sampling of
 metamonads, including <i>C. membranifera</i> and <i>C.
 frisia</i>. Left: matrix of presences (coloured) and absences (light
 grey) of kinetochore, SAC and APC/C proteins that were present in LECA. On
 top: names of the different subunits; single letters (A-X) indicate
 Centromere protein A-X (<i>e.g</i>., CenpA) and numbers, APC/C
 subunit 1-15 (<i>e.g.</i>, Apc1). E2S and E2C, refer to E2
 ubiquitin conjugases S and C, respectively. Colour schemes correspond to
 the kinetochore overview figure on the right and to those used in Figure
 3. Right: cartoon of the components of the kinetochore, SAC signalling,
 the APC/C and its substrates (Cyclin A/B) in LECA and
 <i>Carpediemonas</i> species to indicate the loss of
 components (light grey shading). Blue lines indicate the presence of
 proteins that are part of the MCC. Asterisk: Apc10 has three paralogs in
 <i>C. membranifera</i> and two in <i>C.
 frisia</i>. One is the canonical Apc10, the two others are fused to
 a BTB-Kelch protein of which its closest homologs is a likely adapter for
 the E3 ubiquitin ligase Cullin 3. Supplementary
 Fig. 7: <i>Carpediemonas</i> harbours three different types of
 Histone H3 proteins, a centromere-specific variant (CenpA). Multiple
 sequence alignment of different Histone H3 variants in eukaryotes and
 metamonads, including the secondary structure of canonical H3 in humans
 (pdb: 6ESF_A). CenpA orthologs are characterized by extended amino and
 carboxy termini and a large L1 loop. Red names in the CenpA panel indicate
 for which species centromere/kinetochore localization has been confirmed.
 In addition to CenpA and canonical Histone H3-variants, multiple
 eukaryotes, including <i>C. membranifera</i> and <i>C.
 frisia</i>, harbour other divergent H3 variants. Such divergent
 variants make the annotation of Histone H3 homologs ambiguous (see
 Asterisks; incomplete sequences). Multiple sequence alignments were
 visualized with Jalview<sup>72</sup>, using the Clustal colour
 scheme. Asterisks indicate two potential CenpA candidates in <i>T.
 vaginalis</i> Supplementary
 Fig. 8: Likely presence of SAC signalling in
 <i>Carpediemonas</i>. (a) Short linear motifs form the basis
 of SAC signalling. During prometaphase, unattached kinetochores catalyse
 the production of inhibitor of the cell cycle machinery, a phenomenon
 known as the SAC<sup>73</sup>. (I) The main protein scaffold
 of SAC signalling is the kinase MadBub (paralogs Mad3/Bub1 exist in
 eukaryotes), which consist of many short linear motifs (SLiMs) that
 mediate the interaction of SAC components and the APC/C (light
 blue)<sup>74,75</sup>. MadBub itself is recruited to the
 kinetochore through interaction with Bub3 (GLEBS), which on its turn binds
 repeated phosphomotifs in Knl1<sup>76-78</sup>. The CDI or CMI
 motif aids to recruit Mad1<sup>79-81</sup>, which has a
 Mad2-interaction Motif (MIM) that mediated the kinetochore-dependent
 conversion of open-Mad2 to Mad2 in a closed
 conformation<sup>82</sup>. (II) Mad2, MadBub, Bub3 and 2x
 Cdc20 (APC/C co-activator) form the mitotic checkpoint complex (MCC) and
 block the APC/C<sup>75,83,84</sup>. MadBub contains 3
 different APC/C degrons (D-box, KEN-box and ABBA
 motif)<sup>74</sup> that direct its interaction with 2x Cdc20s
 and effectively make the MCC a pseudo substrate of the APC/C. (III)
 Increasing amounts of kinetochore-microtubule attachments silence the
 production of the MCC at kinetochores and the APC/C is released. Cdc20 now
 presents its substrates Cyclin A and Cyclin B (some eukaryotes have other
 substrates as well, but they are not universally conserved) for
 ubiquitination and subsequent degradation through recognition of a Dbox
 motif<sup>85</sup>. Chromosome segregation will now be
 initiated (anaphase). (b) Presence/absence matrix of motifs involved in
 SAC signalling in a selection of Eukaryotes and Metamonads, including
 <i>C. membranifera</i> and <i>C. frisia</i>.
 Colours correspond to the motifs in panel a, light grey indicates motif
 loss. <i>N</i> signifies the number of MadBub homologs that
 are present in each species. ‘Incomplete’ points to sequences that were
 found to be incomplete due to gaps in the genome assembly. Question marks
 indicate the uncertainty in the presence of that particular motif.
 Although Metamonads have all four MCC components (Mad2, Bub3, MadBub and
 Cdc20), most homologs do not contain the motifs to elicit a canonical SAC
 signalling and it is therefore likely that they do not have a SAC
 response. Exceptions are <i>C membranifera</i>, <i>C.
 frisia</i> and <i>Kipferlia bialata</i>. They retained
 the N-terminal KEN-boxes and one ABBA motif, which are involved in the
 binding of two Cdc20s and a Mad2-interaction motif (MIM) in Mad1 and
 Cdc20. c) Multiple sequence alignments of the motifs from panel A and B.
 Coloured motif boxes correspond to panel a and b. Multiple sequence
 alignments were visualized with Jalview<sup>72</sup>, using
 the Clustal colouring scheme. Asterisks indicate ambiguous motifs in
 <i>Carpediemonas membranifera</i>. Supplementary
 Fig. 9: Histogram showing the frequency distribution of single nucleotide
 variants in the genome of <i>C. membranifera</i>. Diagram
 showing the typical distribution of a haploid genome. Supplementary Fig. 10: Maximum likelihood reconstruction of Endonuclease IV. The unrooted tree contains eukaryotic and prokaryotic Endo IV sequences, showing <i>Carpediemonas</i> sequences emerging within bacterial proteins. The tree was inferred with IQ-TREE under the LG+I+C20 model with 1000 ultrafast bootstraps; alignment length was 276. Scale bar shows the inferred number of amino acid substitutions per site. Supplementary Fig. 11: Maximum likelihood reconstruction of RarA. The unrooted tree contains eukaryotic and prokaryotic sequences, showing <i>Carpediemonas</i> sequences emerging within bacterial proteins. The tree was inferred with IQ-TREE under the LG+I+C20 model with 1000 ultrafast bootstraps; alignment length was 414. Scale bar shows the inferred number of amino acid substitutions per site. Supplementary Fig. 12: Maximum likelihood reconstruction of RNAse H1. <i>Carpediemonas</i> RarA-like proteins emerge within bacterial proteins. Parabasalia and Diplomonada proteins highlighting the proteins have been acquired in different events. The tree was inferred with IQ-TREE under the LG+I+G+C20 model with 1000 ultrafast bootstraps; alignment length was 149. Scale bar shows the inferred number of amino acid substitutions per site.