Protein information

Using ClustalX and ClustalΩ,⁴⁴ each Ciona FA protein sequence was aligned against the human and Xenopus laevis sequence. The sequences were imported into Jalview,⁴⁵ and the most closely aligned regions were isolated. Hydrophobicity plots of each sequence were created using Biopython and code built and modified from Dalke Scientific.⁴⁶ To determine whether the results were significant, the Pearson coefficients were evaluated for the Ciona amino acid (aa) sequence against the human and Xenopus sequences (again using Python), a beta distribution derived for each sequence,^{47 and a} comparison of the critical values to a P < 0.002 level of significance was made. As a standard, P < 0.05 level of significance with 24 tests gives about a 30% chance of a false positive (Type I error), so a more thorough bound of significance was required. The Sidak test,⁴⁸ a familywise error correction method used to reduce type I errors, suggests a P-value of 1 – (1 − 0.05)^1/24, or about 0.0021, where 0.05 is the original level of significance and 24 is the number of comparison tests performed. This assumes that the genes and their products are independent – there does not appear to be any evidence that a mutation in one FA protein leads to the absence of any of the other FA proteins.

Protein structural models (Figs. 2E, F, J, and K and 4F and G) were constructed using Discovery Studio v. 3.1 (BIO-VIA), based on pdb files in the RCSB Protein Data Bank, using 50 iterations with loop refinement. The protein motif diagrams were based on the information in Pfam 29.0.⁴⁹

Analysis of FANCE (A–F) and FANCL (G–K) putative homologs in C. intestinalis. (A) Hydropathy plot of best aligning regions in human, Xenopus, and Ciona putative homologs for FANCE. (B) Best ML tree for alignment of FANCE and putative homologs in C. intestinalis and other eukaryotes. CiUP1 (LOC100186252) has 93% bootstrap support for membership in the clade with vertebrate FANCE proteins. (C) Forcing CiUP1 into the vertebrate FANCE clade does not result in a statistically worse tree, whereas if the locations of the two best C. intestinalis BLAST matches to FANCE are switched in the ML tree (D), the tree is worse at the P < 0.01 level, giving further support to LOC100186252 as the homolog of FANCE. (E,F) Structural modeling of human FANCE and C. intestinalis LOC100186252, showing extreme similarity of overall structures. (G) Hydropathy plot of best aligning regions in human, Xenopus, and Ciona putative homologs for FANCL. (H) Best ML tree for alignment of putative FANCL homologs, showing 87% bootstrap support for Cifancl clustering with vertebrate and other FANCL proteins. (I) Diagrammatic comparison of human and C. intestinalis FANCL inferred protein motifs. (J,K) Modeling of D. melanogaster and C. intestinalis FANCL protein structures.

Analysis of FANCD2 (A–G) and FANCI (H–K) putative homologs in C. intestinalis. (A) Hydropathy plot of best aligning regions in human, Xenopus, and Ciona putative homologs for FANCD2. (B) Best ML tree for alignment of FANCE and putative homologs in C. intestinalis and other eukaryotes. Cifancd2 groups closely with FANCD2, but with low bootstrap support. (C) If Cifancd2 is moved out of the FANCD2 clade, the tree is statistically worse at the P < 0.02 level, supporting the case for Cifancd2 as a true homolog of FANCD2. (D) Alignment of human, mouse, and C. intestinalis FANCD2 protein sequences showing conservation of L215, P216, L234, and L235, critical residues of the CUE domain (red boxes).⁶³ (E) Alignment showing partial conservation of critical residues around human aa 525 (arrows, and box), as well as K561, the site of monoubiquitination⁵ (red arrowhead) in C. intestinalis. (F, G) Modeling of mouse and C. intestinalis FANCD2 homolog protein structures, respectively. (H) Hydropathy plot of best aligning regions in human, Xenopus, and Ciona putative homologs for FANCI. (I) Best ML tree for alignment of putative FANCI homologs, showing weak bootstrap support for Cifancl being more closely related to FANCI than the next most similar C. intestinalis protein. (J) Alignment of human, mouse, and C. intestinalis FANCI protein sequences showing the conservation of K523 and K715, the site of FANCI monoubiquitination,^6,7 and, the site of FANCI SUMOylation, respectively (arrowheads). (K) SQ/TQ phosphosite clusters (red boxes) shown to be critical for FANCI function.⁶⁸ Dashed boxes denote possible additional functional SQ/TQ phosphosite clusters in C. intestinalis sequence not found in humans and mouse.