Dimerization of Cdc13 is essential for dynamic DNA exchange on telomeric DNA

Abstract

Eukaryotic single-stranded DNA (ssDNA)-binding proteins (ssBPs) protect telomeres from nuclease activity. In Saccharomyces cerevisiae, the ssBP Cdc13 is an essential protein that acts as a central regulator of telomere length homeostasis and chromosome end protection. Cdc13 has high binding affinity for telomeric ssDNA, with a very slow off-rate. Previously, we reported that despite this tight ssDNA binding, Cdc13 rapidly exchanges between bound and unbound telomeric ssDNA substrates, even at sub-stoichiometric concentrations of competitor ssDNA. This dynamic DNA exchange (DDE) is dependent on the presence and length of telomeric repeat sequence ssDNA and requires both Cdc13 DNA binding domains, OB1 and OB3. Here, we investigated if Cdc13 dimerization is important for DDE by characterizing the dimerization mutant Cdc13-L91R. Using mass photometry, we confirmed that Cdc13-L91R fails to dimerize in solution, even in the presence of ssDNA. DDE assays revealed that Cdc13-L91R fails to undergo ssDNA exchange compared to the recombinant wild-type protein. Biolayer interferometry demonstrated that this effect was not due to differences in ssDNA binding kinetics. Thus, dimerization of Cdc13 is essential for DDE, and we model how this may impact telomere biology in vivo.

Keywords: Cdc13; DNA-binding protein; DNA–protein interaction; OB fold; Saccharomyces cerevisiae; dynamic DNA exchange; molecular biology; telomere.

Figure 1

Mass photometry of WT Cdc13. A, recombinant Cdc13 exists as both monomers and dimers in PBS. B, pre-binding Cdc13 to Tel30G ssDNA yields only dimers. C, only Cdc13 dimers are observed in 1× binding buffer. D, the Tel50G substrate can be bound by two Cdc13 dimers. All assays contained 11.25 nM protein and 50 nM ssDNA, where indicated. The data shown are representative of ≥3 independent experiments.

Figure 2

Cdc13-L91R is monomeric is solution.A, recombinant Cdc13-L91R exists as a monomer in solution, and ssDNA binding (Tel30G) does not induce dimerization (B). C, Tel50G supports the binding of two Cdc13-L91R monomers. All assays were conducted as in Figure 1.

Figure 3

Cdc13-L91R lacks DDE activity.A, representative gel of the dissociation of 3.75 nM WT Cdc13 dimer prebound to 2 nM IR800-Tel30G and exposed to increasing concentrations (0.2–15 nM) of IR700-Tel30G. B, representative gel of the dissociation of 3.75 nM Cdc13-L91R monomer prebound to 2 nM IR800-Tel30G and exposed to increasing concentrations of IR700-Tel30G. DNA migrating slower than the protein-ssDNA complex is also evident in this gel. Its identity is unknown but could be two monomers of Cdc13-L91R bound to a single substrate, or evidence that Tel30G forms intermolecular G-quadruplexes or other secondary structures. C, plot of the displacement of the initially bound IR800-Tel30G DNA by IR700-Tel30G. Error bars are the standard deviation of the mean of ≥3 replicates.

Figure 4

BLI analysis of Cdc13 and Cdc13-L91R binding to telomeric ssDNA.A, association of 100 nM Cdc13 or Cdc13-L91R with immobilized Tel30G ssDNA was monitored for 2 min. B, Protein-ssDNA complexes were diluted into a large volume of DNA-free buffer, and dissociation was monitored for 2 min. C, the remaining protein-ssDNA complexes were exposed to free unlabeled Tel30G ssDNA in solution, driving either DDE (Cdc13) or simple binding competition (Cdc13-L91R). The curves shown are comprised of data points collected every 0.2 s and are representative of ≥3 independent experiments. The Cdc13 data are from (9) and were collected at the same time as the Cdc13-L91R data.

Figure 5

Cdc13 dimer models for DDE. Cdc13 homodimers contain four ssDNA binding sites: the OB1 and OB3 domains from each subunit. A long ssDNA substrate (top, red) can fill both OB1 and OB3 sites in one monomer (left), fill both high-affinity OB3 sites across the dimer (middle), or span the OB1 domain of subunit ‘A’ in the dimer and the OB3 domain of subunit ‘B’ (or vice versa). All three scenarios leave two open ssDNA binding sites that can be filled when encountering a secondary ssDNA substrate (blue, middle). Upon filling the second set of binding sites, the binding event is allosterically communicated across the dimer, leading to release of the primary ssDNA to complete the dynamic exchange of substrates (DDE, bottom). These models are depicted with two separate ssDNAs, but a single substrate of sufficient length, especially one that is actively being lengthened by telomerase, is predicted to fulfill the same role.