Dynamic DNA Shortening by Telomere-Binding Protein Cdc13

Abstract

Telomeres are essential for chromosome maintenance. Cdc13 is a single-stranded telomeric DNA binding protein that caps telomeres and regulates telomerase function in yeast. Although specific binding of Cdc13 to telomeric DNA is critical for telomere protection, the detail mechanism how Cdc13-DNA complex protects telomere is unclear. Using two single-molecule methods, tethered particle motion and atomic force microscopy, we demonstrate that specific binding of Cdc13 on single-stranded telomeric DNA shortens duplex DNA into distinct states differed by ∼70−80 base pairs. DNA shortening by Cdc13 is dynamic and independent of duplex DNA sequences or length. Significantly, we found that Pif1 helicase is incapable of removing Cdc13 from the shortened DNA-Cdc13 complex, suggesting that Cdc13 forms structurally stable complex by shortening of the bound DNA. Together our data identified shortening of DNA by Cdc13 and provided an indication for efficient protection of telomere ends by the shortened DNA-Cdc13 complex.

Molecular architecture and oligomerization of Candida glabrata Cdc13 underpin its telomeric DNA-binding and unfolding activity

“…In addition, we find that OB1 induces the formation of large oligomers of Cdc13 formed by the stepwise addition of Cdc13 dimers, and we have mapped residues required for OB1 oligomerization using mutational analysis. Our data is consistent with previous findings on the oligomerization of yeast Cdc13 ( 24 , 30 , 34 ) and human CST ( 21 ). Thus, we speculate that the ability of OB1 to direct the interaction of several Cdc13 dimers may play a role in altering the structure of ssDNA.…”

“…These observations reflect the ability of Cdc13 to interact with other Cdc13 molecules to form larger complexes when bound to ssDNA. Whether this is a bona-fide activity, as suggested for S. cerevisiae Cdc13 ( 24 , 30 , 34 ), or a consequence of the recombinant overexpression of the protein is something we cannot fully determine with our experiments (see experiments and discussions below).…”

“…Finally, Lin et al. found that S. cerevisiae Cdc13 can remodel telomeric DNA suggesting the formation of a nucleoprotein complex based on the oligomerization of Cdc13 ( 30 ). Our single-molecule experiments show that OB1 is needed for the full maintenance of an unfolded state of ssDNA bound to Cdc13, which would fit a model where OB1 facilitates the oligomerization of Cdc13 on the DNA.…”

“…When Flag-tagged and V5-tagged CTC1 are co-expressed, either tag immunoprecipitates the other and this has been interpreted as proof that higher-order CST oligomeric species form in human cells and could have functional significance ( 21 ). Formation of oligomeric Cdc13 complexes have also been suggested to explain how S. cerevisiae Cdc13 shortened DNA in experiments using single-molecule methods ( 30 ).…”

A comprehensive map of hotspots of de novo telomere addition inSaccharomyces cerevisiae

“…Rap1 participates in telomere length homeostasis, telomere capping, and formation of telomeric chromatin (Hardy et al 1992; Kyrion et al 1993; Marcand et al 1997; Negrini et al 2007; Pardo & Marcand, 2005; Teixeira et al 2004; Vodenicharov et al 2010), while Cdc13 interacts with the Est1 component of telomerase to recruit telomerase to telomeres (Evans & Lundblad, 1999; Pennock et al 2001, Chen et al 2018). Cdc13 additionally interacts with Stn1 and Ten1 to limit nucleolytic resection and promote fill-in synthesis by the lagging strand polymerase machinery (Pennock et al 2001; Lin et al 2021).…”