The tenacious recognition of yeast telomere sequence by Cdc13 is fully exerted by a single OB-fold domain

Abstract

Cdc13, the telomere end-binding protein from Saccharomyces cerevisiae, is a multidomain protein that specifically binds telomeric single-stranded DNA (ssDNA) with exquisitely high affinity to coordinate telomere maintenance. Recent structural and genetic data have led to the proposal that Cdc13 is the paralog of RPA70 within a telomere-specific RPA complex. Our understanding of Cdc13 structure and biochemistry has been largely restricted to studies of individual domains, precluding analysis of how each domain influences the activity of the others. To better facilitate a comparison to RPA70, we evaluated the ssDNA binding of full-length S. cerevisiae Cdc13 to its minimal substrate, Tel11. We found that, unlike RPA70 and the other known telomere end-binding proteins, the core Cdc13 ssDNA-binding activity is wholly contained within a single tight-binding oligosaccharide/oligonucleotide/oligopeptide binding (OB)-fold. Because two OB-folds are implicated in dimerization, we also evaluated the relationship between dimerization and ssDNA-binding activity and found that the two activities are independent. We also find that Cdc13 binding exhibits positive cooperativity that is independent of dimerization. This study reveals that, while Cdc13 and RPA70 share similar domain topologies, the corresponding domains have evolved different and specialized functions.

Human CST Prefers G-Rich but Not Necessarily Telomeric Sequences

“…The specificity-determining elements are distributed rather evenly across the oligonucleotide, in contrast to the 5’-preference observed with the telomere-binding proteins of S. cerevisiae Cdc13 and hPOT1. 8,14,15 …”

“…These results suggest that the specific mode of the ssDNA-binding interface of the CST complex can accommodate a more diverse range of G-rich sequences than other telomere end-binding proteins can. 8,15,51 …”

“…Extensive structural and biochemical evidence revealed that the Saccharomyces cerevisiae Cdc13-Stn1-Ten1 complex contains a domain architecture that parallels that of RPA;11,12 however, the Cdc13 component possesses tight picomolar affinity and sequence specificity uniquely suited to the particular heterogeneity exhibited by yeast telomeres 11,13–15. This specificity, however, is not widely retained even within Saccharomycotina.…”

Tying up the Ends: Plasticity in the Recognition of Single-Stranded DNA at Telomeres

“…116,117,123–128 While Cdc13 exists as a homodimer in vitro , 118 disruption of the dimer with a single point mutation at L91R does not impact ssDNA-binding activity, as the monomeric form of Cdc13 binds the 11-nt sequence with similarly tight affinity. 128 Interestingly, Cdc13 binds longer strands of telomeric ssDNA with modest positive cooperativity. This activity is independent of its ability to dimerize, suggesting instead that the binding of Cdc13 to a G-rich strand alters the conformation of the nucleic acid such that it is more conducive for binding a second Cdc13 protein.…”

“…116,123,124,126,128 The solution structure of this complex revealed that the ssDNA binds by stretching out across a large binding surface to accommodate the extended 11-nt sequence (Figure 9). 116,117 Residues that interact with the DNA are predominantly aromatic or basic.…”