Abstract:The human cancer susceptibility gene, BRCA2, functions in double-strand break repair by homologous recombination, and it appears to function via interaction of a repetitive region ("BRC repeats") with RAD-51. A putatively simpler homolog, dmbrca2, was identified in Drosophila melanogaster recently and also affects mitotic and meiotic double-strand break repair. In this study, we examined patterns of repeat variation both within Drosophila pseudoobscura and among available Drosophila genome sequences. We identi… Show more
“…Similar patterns of rapid repeat evolution have been observed for proteins involved in adaptive evolution, for example in VERL, a protein involved in egg-sperm interaction in abalones ( Panhuis et al 2006 ), in PRDM9, a protein involved in homologous recombination during meiosis ( Oliver et al 2009 ), and in the arms race between zinc-finger proteins and retrotransposons ( Jacobs et al 2014 ). Repeats in some core cellular proteins such as structural BRC repeats in the DNA-damage-related protein BRCA2 ( Bennett and Noor 2009 ; Lou et al 2014 ) and a phosphomotif in the C-terminal domain of RNA polymerase ( Chunlin Yang and Stiller 2014 ) have likewise undergone striking repeat evolution in specific clades. To our knowledge however, our study is the first to trace such extensive dynamic repeat evolution for a disordered signaling protein across all eukaryotic supergroups.…”
The outer kinetochore protein scaffold KNL1 is essential for error-free chromosome segregation during mitosis and meiosis. A critical feature of KNL1 is an array of repeats containing MELT-like motifs. When phosphorylated, these motifs form docking sites for the BUB1–BUB3 dimer that regulates chromosome biorientation and the spindle assembly checkpoint. KNL1 homologs are strikingly different in both the amount and sequence of repeats they harbor. We used sensitive repeat discovery and evolutionary reconstruction to show that the KNL1 repeat arrays have undergone extensive, often species-specific array reorganization through iterative cycles of higher order multiplication in conjunction with rapid sequence diversification. The number of repeats per array ranges from none in flowering plants up to approximately 35–40 in drosophilids. Remarkably, closely related drosophilid species have independently expanded specific repeats, indicating near complete array replacement after only approximately 25–40 Myr of evolution. We further show that repeat sequences were altered by the parallel emergence/loss of various short linear motifs, including phosphosites, which supplement the MELT-like motif, signifying modular repeat evolution. These observations point to widespread recurrent episodes of concerted KNL1 repeat evolution in all eukaryotic supergroups. We discuss our findings in the light of the conserved function of KNL1 repeats in localizing the BUB1–BUB3 dimer and its role in chromosome segregation.
“…Similar patterns of rapid repeat evolution have been observed for proteins involved in adaptive evolution, for example in VERL, a protein involved in egg-sperm interaction in abalones ( Panhuis et al 2006 ), in PRDM9, a protein involved in homologous recombination during meiosis ( Oliver et al 2009 ), and in the arms race between zinc-finger proteins and retrotransposons ( Jacobs et al 2014 ). Repeats in some core cellular proteins such as structural BRC repeats in the DNA-damage-related protein BRCA2 ( Bennett and Noor 2009 ; Lou et al 2014 ) and a phosphomotif in the C-terminal domain of RNA polymerase ( Chunlin Yang and Stiller 2014 ) have likewise undergone striking repeat evolution in specific clades. To our knowledge however, our study is the first to trace such extensive dynamic repeat evolution for a disordered signaling protein across all eukaryotic supergroups.…”
The outer kinetochore protein scaffold KNL1 is essential for error-free chromosome segregation during mitosis and meiosis. A critical feature of KNL1 is an array of repeats containing MELT-like motifs. When phosphorylated, these motifs form docking sites for the BUB1–BUB3 dimer that regulates chromosome biorientation and the spindle assembly checkpoint. KNL1 homologs are strikingly different in both the amount and sequence of repeats they harbor. We used sensitive repeat discovery and evolutionary reconstruction to show that the KNL1 repeat arrays have undergone extensive, often species-specific array reorganization through iterative cycles of higher order multiplication in conjunction with rapid sequence diversification. The number of repeats per array ranges from none in flowering plants up to approximately 35–40 in drosophilids. Remarkably, closely related drosophilid species have independently expanded specific repeats, indicating near complete array replacement after only approximately 25–40 Myr of evolution. We further show that repeat sequences were altered by the parallel emergence/loss of various short linear motifs, including phosphosites, which supplement the MELT-like motif, signifying modular repeat evolution. These observations point to widespread recurrent episodes of concerted KNL1 repeat evolution in all eukaryotic supergroups. We discuss our findings in the light of the conserved function of KNL1 repeats in localizing the BUB1–BUB3 dimer and its role in chromosome segregation.
“…The arrangement of the BRC repeats in T. brucei BRCA2 is also unusual: most of the repeats are identical in sequence and all form a tandem array (27). This contrasts with BRCA2 in humans (Supplementary Figure S1), plants (11) and at least some Drosophila species (29), where the BRC repeats are variable in sequence and dispersed throughout the polypeptide. Figure 1.Growth and repair efficiency of T. brucei BRCA2 mutants.…”
Trypanosoma brucei survives in mammals through antigenic variation, which is driven by RAD51-directed homologous recombination of Variant Surface Glycoproteins (VSG) genes, most of which reside in a subtelomeric repository of >1000 silent genes. A key regulator of RAD51 is BRCA2, which in T. brucei contains a dramatic expansion of a motif that mediates interaction with RAD51, termed the BRC repeats. BRCA2 mutants were made in both tsetse fly-derived and mammal-derived T. brucei, and we show that BRCA2 loss has less impact on the health of the former. In addition, we find that genome instability, a hallmark of BRCA2 loss in other organisms, is only seen in mammal-derived T. brucei. By generating cells expressing BRCA2 variants with altered BRC repeat numbers, we show that the BRC repeat expansion is crucial for RAD51 subnuclear dynamics after DNA damage. Finally, we document surprisingly limited co-localization of BRCA2 and RAD51 in the T. brucei nucleus, and we show that BRCA2 mutants display aberrant cell division, revealing a function distinct from BRC-mediated RAD51 interaction. We propose that BRCA2 acts to maintain the huge VSG repository of T. brucei, and this function has necessitated the evolution of extensive RAD51 interaction via the BRC repeats, allowing re-localization of the recombinase to general genome damage when needed.
“…The variability in the number of BRC repeats even within the
Drosophila species has provided fodder for further speculation on the evolution of this gene
17,
20 . The detection of a new BRC repeat would necessitate the reevaluation of such hypotheses.…”
Mutations in the BRCA2 tumor suppressor protein leave individuals susceptible to breast, ovarian and other cancers. The BRCA2 protein is a critical component of the DNA repair pathways in eukaryotes, and also plays an integral role in fostering genomic variability through meiotic recombination. Although present in many eukaryotes, as a whole the
BRCA2 gene is weakly conserved. Conserved fragments of 30 amino acids (BRC repeats), which mediate interactions with the recombinase RAD51, helped detect orthologs of this protein in other organisms. The carboxy-terminal of the human BRCA2 has been shown to be phosphorylated by checkpoint kinases (Chk1/Chk2) at T3387, which regulate the sequestration of RAD51 on DNA damage. However, apart from three BRC repeats, the
Drosophila melanogaster gene has not been annotated and associated with other functionally relevant sequence fragments in human BRCA2. In the current work, the carboxy-terminal phosphorylation threonine site (E=9.1e-4) and a new BRC repeat (E=17e-4) in
D. melanogaster has been identified, using a fragmented alignment methodology (FRAGAL). In a similar study, FRAGAL has also identified a novel half-a- tetratricopeptide (HAT) motif (E=11e-4), a helical repeat motif implicated in various aspects of RNA metabolism, in Utp6 from yeast. The characteristic three aromatic residues with conserved spacing are observed in this new HAT repeat, further strengthening my claim. The reference and target sequences are sliced into overlapping fragments of equal parameterized lengths. All pairs of fragments in the reference and target proteins are aligned, and the gap penalties are adjusted to discourage gaps in the middle of the alignment. The results of the best matches are sorted based on differing criteria to aid the detection of known and putative sequences. The source code for FRAGAL results on these sequences is available at
https://github.com/sanchak/FragalCode, while the database can be accessed at
www.sanchak.com/fragal.htm
l.
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