Hypothesis: Paralog Formation from Progenitor Proteins and Paralog Mutagenesis Spur the Rapid Evolution of Telomere Binding Proteins

Abstract

Through elegant studies in fungal cells and complex organisms, we propose a unifying paradigm for the rapid evolution of telomere binding proteins (TBPs) that associate with either (or both) telomeric DNA and telomeric proteins. TBPs protect and regulate telomere structure and function. Four critical factors are involved. First, TBPs that commonly bind to telomeric DNA include the c-Myb binding proteins, OB-fold single-stranded binding proteins, and G-G base paired Hoogsteen structure (G4) binding proteins. Each contributes independently or, in some cases, cooperatively, to provide a minimum level of telomere function. As a result of these minimal requirements and the great abundance of homologs of these motifs in the proteome, DNA telomere-binding activity may be generated more easily than expected. Second, telomere dysfunction gives rise to genome instability, through the elevation of recombination rates, genome ploidy, and the frequency of gene mutations. The formation of paralogs that diverge from their progenitor proteins ultimately can form a high frequency of altered TBPs with altered functions. Third, TBPs that assemble into complexes (e.g., mammalian shelterin) derive benefits from the novel emergent functions. Fourth, a limiting factor in the evolution of TBP complexes is the formation of mutually compatible interaction surfaces amongst the TBPs. These factors may have different degrees of importance in the evolution of different phyla, illustrated by the apparently simpler telomeres in complex plants. Selective pressures that can utilize the mechanisms of paralog formation and mutagenesis to drive TBP evolution along routes dependent on the requisite physiologic changes.

Keywords: evolution; models; non-LTR reverse transcription; stress response; telomerase; telomeres.

FIGURE 1

Paralog formation and mutagenesis of a single ORF1. Under conditions of stress response and high selectivity, recombination and mutagenesis increase the frequency of paralog formation during evolution. In this process, recombination results in duplication of the ORF1 coding sequence. The first copy, when separated by recombination, remains stable as ORF1. Under selection, the paralog undergoes an elevated level of mutagenesis caused by stress in response to dysfunctional telomeres. Unknown multiple rounds of mutagenesis take place in evolutionary time to ultimately give rise to a unique functional protein, ORF2. In the ORC1 example, paralog formation gives rise to Sir3, a protein involved in silencing of genes and the structure of telomeres in several yeast strains.

FIGURE 2

Minimal function cis-acting elements at the telomere. A minimal telomere consists of different combinations of modular domains. (A) MR, referring to the MRX and MRN (duplex binding) complexes and the CTC complex that binds to single-stranded are required telomeric DNA binding proteins that are common to the DNA of most telomeres. Both proteins will result in far greater stability. (B) In the second module, the DNA duplex binding is mediated by c-myb binding sequences. TBP will also bind to G4DNA structures. Of course combinations of these modules are possible and in both cases MR binding to duplex DNA and the Cst1/Stn1/Ten1 (CST) binding to single stranded DNA will increase stability. Green indicates c-myb. This model is similar to the one proposed by Linger and Price (2009). Each modular protein can mutate or recombine with other modules to give rise to the possibility of modular based mutagenesis. (C) In plants, a different module is found. A single essential structure is formed that contains the c-myb domain with histone H1 folds to bind duplex DNA. Also, TBP associates with G4DNA. Other proteins that are present in plants and bind to ends include the MR and CTC complexes as in animal and fungal species. Ultimately the MR structure provides equilibrium of telomere sizes that serve to protect the end from double-strand break processing enzymes, resulting in the anti-checkpoint.

FIGURE 3

Domain structure of Orc1 and Sir3. The paralog, SIR3, shares the highly conserved Bromo adjacent homology (BAH) domain associated with transcriptional regulation, the AAA-type ATPase, and Cdc6 site. The sequence homology of ORC1 ends at amino acid 738 out of 914 residues. The Orc1 gaps are amino acids that differ between Orc1 and Sir3. The CTD of Sir3 (amino acids 820–978) is predicted to have a winged helical structure similar to Cdc6. The winged helix motif plays a role in DNA binding and protein/binding recognition, including the associations of histones H3 and H4. The numbered amino acid sequence refers to Orc1 amino acid number.

FIGURE 4

The fungal phylogenetic tree shows the two pathogenic species. On the left is shown the phylogenetic map for fungi showing the point of WGD for clarity. On the right is a tree rooted in similarity to S. cerevisiae Sir3 that is discussed in the text. Green indicates the S. cerevisiae Sir3 and S. byanus. Two lines below are depicted by the orange star are the Candida glabrata and Nakaseomyces delphensis strains. All strains are part of the ancestral WGD.

FIGURE 5

Sequence Homology of CTD (CTT) in differing Saccharomyces species. The lines (from top to bottom) display Sir3 from Saccharomyces cerevisiae sequence, S. byanus, S. milkatae a, S. paradoxis, an independent S. byanus, S. castilli, and S. kudriavzevii. Yellow refers to identity, pink to high similarity, and green to statistical similarity.

FIGURE 6

Proteomic view of Association of Cdc6 with both Orc1 and Sir3 Protein-based associations are present between Orc1 and other yeast nuclear factors. We conducted an SGD search for physical interactions between Orc1 or Sir3 and other cellular proteins using at least four experiments. Orc1 is capable of associating with Cdc6 while Sir3 is not. In no experiment was Cdc6/Sir3 binding observed. One genetic interaction between Orc3 and Orc6 is also shown in this figure.

FIGURE 7

High sequence similarity is present between C. glabrata (Cg) and N. delphensis (Nr). The two different CTD sequences are shown in two close pathogenic species related to Candida. Red nucleotides shown identity and orange residues show similarities.

FIGURE 8

Selected stress responses and surface interactions. Selective Stress (A–D) shows the proposed effect of stress responses over evolutionary time. (A) The initial modular telomere with a TBP (red). (B) Duplication of DNA (orange) encoding the protein. (C) Separation and mutagenesis (purple) of the two DNAs and the altered protein (orange and purple box. (D) The final telomere, bound by the mutated protein. Protein Legend: c-myb containing protein: green, G4 DNA binding protein (star), and OB-fold protein (yellow). Protein interfaces. In example (E), protein (blue) associated with the c-myb protein with an unfavorable surface interaction shown by the x. (F) Protein interfaces that interact favorably with a second protein (red) to form a stable structure as indicated by the +. A simplified minimal modular telomere is shown just for reference.