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bioRxiv · 10.1101/2025.03.25.645294

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

Nickens, D. G.·Gray, S. J.·Simmons, R. H.·Bochman, M. L.
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Abstract

Single-stranded DNA (ssDNA) binding proteins (ssBPs) are essential in eukaryotes to 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, both alone and as part of the Cdc13-Stn1-Ten1 (CST) complex. 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. Gel-based DDE assays revealed that Cdc13-L91R fails to undergo ssDNA exchange compared to 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. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/645294v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@18f34fdorg.highwire.dtl.DTLVardef@e00e71org.highwire.dtl.DTLVardef@1d4400aorg.highwire.dtl.DTLVardef@1b87a7e_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Nickens, D. G., Gray, S. J., Simmons, R. H., Bochman, M. L.. 2025-03-26. Dimerization of Cdc13 is essential for dynamic DNA exchange on telomeric DNA. https://doi.org/10.1101/2025.03.25.645294

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