Evolution of CST function in telomere maintenance

Abstract

Telomeres consist of an elaborate, higher-order DNA architecture, and a suite of proteins that provide protection for the chromosome terminus by blocking inappropriate recombination and nucleolytic attack. In addition, telomeres facilitate telomeric DNA replication by physical interactions with telomerase and the lagging strand replication machinery. The prevailing view has been that two distinct telomere capping complexes evolved, shelterin in vertebrates and a trimeric complex comprised of Cdc13, Stn1 and Ten1 (CST) in yeast. The recent discovery of a CST-like complex in plants and humans raises new questions about the composition of telomeres and their regulatory mechanisms in multicellular eukaryotes. In this review we discuss the evolving functions and interactions of CST components and their contributions to chromosome end protection and DNA replication.

Figure 1

Telomere capping complexes in vertebrates and yeast. (A) The six-member shelterin complex associates with both single- and double-strand regions of the vertebrate telomeric DNA. (B) Budding yeast telomeres are protected by the trimeric Cdc13 Stn1 Ten1 (CST) complex, which assembles on the G-overhang. The duplex region of the telomere is bound by a separate complex containing Rap1, Rif1 and Rif2. (C) Fission yeast telomeres associate with a six member shelterin-like complex. In addition, Stn1 and Ten1 contribute to chromosome end protection, but it is not known how they interact with other telomere proteins.

Figure 2

Similar domain structure in CST and RPA. The predicted domain structure is shown for CTC1 from humans and Arabidopsis and STN1 and TEN1 from humans, Arabidopsis, S. pombe and S. cerevisiae. For comparison, human RPA structure is shown.

Figure 3

Stn1 and Rpa32 cluster in distinct monophyletic groups. Shown is an unrooted maximum likelihood phylogeny of the OB-fold domains of STN1 and RPA32 inferred using the WAG amino-acid transition model in RAxML from the alignment of Gao et al. with the addition of STN1 from plants and green algae. Numbers along branches are bootstrap percentages from 500 replicates and indicate that STN1 and RPA32 form distinct monophyletic groups. Arrows indicate the placement of Arabidopsis. Other species are: Ag, Ashbya gossypii; An, Aspergillus nidulans; Cg, Candida glabrata; Dh, Debaryomyces hansenii; Dr, Danio rerio; Gg, Gallus gallus; Gz, Gibberella zeae; Hs, Homo sapiens; Kl, Kluyveromyces lactis; Nc, Neurospora crassa; Mm, Mus musculus; Os, Oryza sativa; Ol, Ostreococcus lucimarinus; Sc, Saccharomyces cerevisiae; Xt, Xenopus tropicalis.

Figure 4

Arabidopsis CTC1 interacts with the catalytic subunit of DNA polymerase α (ICU2) in vitro. (A) Diagram of DNA polymerase α domain structure. (B) Co-immunopreciptiation was conducted with ³⁵S-Met labeled (asterisk) and T7-tagged unlabeled protein expressed in rabbit reticulocyte lysate. The C-terminal half of AtCTC1 was used for binding reactions with different regions of ICU2. When bound to a tagged partner, labeled protein is precipitated on T7-beads (b) from the unbound supernatant (u) fraction. Ku70/80 interaction served as a positive control.

Figure 5

Model for telomere capping complexes in the flowering plant Arabidopsis thaliana and the moss Physcomitrella patens. (A) CST functions as the major telomere capping complex in Arabidopsis. Multiple TRF-like proteins have been described, but other shelterin-like components cannot be identified in plant genomes. POT1a is a telomerase accessory factor and is not required for chromosome end protection. (B) P. patens encodes two TRF-like proteins and a single POT1 protein. The moss POT1 binds single-stranded G-rich telomeric DNA and functions in a manner analogous to vertebrate and yeast POT1. CST components are encoded in the P. patens genome, but their function is unknown.

Figure 6

Knockdown of human CST components, CTC1 or STN1, results in multi-telomeric signals (MTS). (A) Examples of MTS in stable shRNA knockdown clones of either CTC1 or STN1. Telomeric PNA-FITC probe (green); DAPI (blue). (B) Quantification of MTS. Black and gray bars represent independent experiments.

Figure 7

Model for CST in telomeric DNA replication in budding yeast and vertebrates. S. cerevisiae CST interacts with the Est1 component of telomerase to promote telomeric DNA synthesis on the G-overhang, and with Polα/primase to facilitate lagging strand replication of the C-strand. Vertebrate CST associates with Polα/primase and stimulates its priming activity. The shelterin component TPP1 contacts telomerase and is postulated to recruit it to the chromosome terminus. TPP1 may also recruit CST to the telomere via interactions with STN1.

Figure 8

Model for CST function during replication of non-telomeric DNA. Replication stress (following DNA damage or synthesis through highly repetitive sequences) results in polymerase dissociation from replicative helicases. CST may recruit and stimulate the activity of DNA polα/primase to promote lagging strand replication at such sites.