Single-stranded telomere-binding protein employs a dual rheostat for binding affinity and specificity that drives function Leslie W. Glustroma,1, Kenneth R. Lyona,1,2, Margherita Paschinib,c,3, Cynthia M. Reyesb,c, Nicholas V. Parsonneta, Tasha B. Torob,4, Victoria Lundbladb,c, and Deborah S. Wuttkea,5 aDepartment of Biochemistry, University of Colorado, Boulder, CO 80309; bSalk Institute for Biological Studies, La Jolla, CA 92037; and cDivision of Biological Sciences, University of California, San Diego, La Jolla, CA 92093 Edited by Juli Feigon, University of California, Los Angeles, CA, and approved August 28, 2018 (received for review January 17, 2018) ssDNA, which is involved in numerous aspects of chromosome the genome, RPA also needs to bind ssDNA indiscriminately. biology, is managed by a suite of proteins with tailored activities. Commensurate with this expectation, RPA displays little obvi- The majority of these proteins bind ssDNA indiscriminately, exhib- ous sequence preference in vitro, binding ssDNA tenaciously iting little apparent sequence preference. However, there are with single-digit nanomolar affinities (6, 7). several notable exceptions, including the Saccharomyces cerevisiae In contrast to the genome-wide and the apparently sequence- Cdc13 protein, which is vital for yeast telomere maintenance. Cdc13 nonspecific role performed by the canonical RPA complex (2), is one of the tightest known binders of ssDNA and is specific for G- proteins that interact with ssDNA overhangs at telomeres exhibit – rich telomeric sequences. To investigate how these two different sequence specificity tuned to the G-rich telomeric repeats (9 11). biochemical features, affinity and specificity, contribute to function, These telomere-dedicated proteins also show exceptional affini- we created an unbiased panel of alanine mutations across the ties for their ssDNA ligands, ranging from the tight nanomolar binding by human Pot1 to single-digit picomolar binding by the Cdc13 DNA-binding interface, including several aromatic amino acids Saccharomyces cerevisiae that play critical roles in binding activity. A subset of mutant pro- Cdc13 protein (9, 11, 12). Remarkably, the Cdc13 protein performs its telomere-dedicated role as a teins exhibited significant loss in affinity in vitro that, as expected, BIOCHEMISTRY conferred a profound loss of viability in vivo. Unexpectedly, a sec- subunit of a heterotrimeric complex with a domain architecture that closely parallels that of RPA (13). In both the canonical and ond category of mutant proteins displayed an increase in specificity, telomere-dedicated RPA, the large subunit is constitutively asso- manifested as an inability to accommodate changes in ssDNA se- ciated with two smaller proteins, Stn1/Ten1 with Cdc13 and quence. Yeast strains with specificity-enhanced mutations displayed Rpa32/Rpa14 with RPA70. In the CST complex, high affinity for a gradient of viability in vivo that paralleled the loss in sequence ssDNA is conferred by the large subunit, whereas in RPA high tolerance in vitro, arguing that binding specificity can be fine-tuned to ensure optimal function. We propose that DNA binding by Cdc13 employs a highly cooperative interface whereby sequence diversity Significance is accommodated through plastic binding modes. This suggests that sequence specificity is not a binary choice but rather is a continuum. Proteins that bind nucleic acids are frequently categorized as Even in proteins that are thought to be specific nucleic acid binders, being either specific or nonspecific, with interfaces to match sequence tolerance through the utilization of multiple binding that activity. In this study, we have found that a telomere- modes may be a broader phenomenon than previously appreciated. binding protein exhibits a degree of specificity for ssDNA that is finely tuned for its function, which includes specificity for G- Cdc13 | DNA-binding specificity | telomere maintenance | single-stranded rich sequences with some tolerance for substitution. Mutations DNA binding | t-RPA of the protein that dramatically impact its affinity for single- stranded telomeric DNA are lethal, as expected; however, mutations that alter specificity also impact biological function. he proper management of ssDNA in the cell is required for Unexpectedly, we found mutations that make the protein numerous aspects of chromosome biology. In all kingdoms of T more specific are also deleterious, suggesting that specificity life, ssDNA is formed transiently during the execution of many and nonspecificity in nucleic acid recognition may be achieved essential cellular processes including transcription, DNA replica- through more nuanced mechanisms than currently recognized. tion, recombination, and repair. To coordinate these numerous activities, a diverse array of proteins has evolved to bind ssDNA, Author contributions: V.L. and D.S.W. designed research; L.W.G., K.R.L., M.P., C.M.R., to facilitate normal events such as DNA replication, or to signal N.V.P., and T.B.T. performed research; L.W.G., K.R.L., M.P., C.M.R., and N.V.P. analyzed the appearance of inappropriate ssDNA and initiate repair (1). data; and V.L. and D.S.W. wrote the paper. Several of these ssDNA-binding proteins function in genome- The authors declare no conflict of interest. wide maintenance (1, 2). Widely studied examples include the This article is a PNAS Direct Submission. bacterial single-strand–binding protein (SSB) and its functional This open access article is distributed under Creative Commons Attribution-NonCommercial- equivalent in eukaryotes, replication protein A (RPA) (3–5). SSB NoDerivatives License 4.0 (CC BY-NC-ND). and RPA are both essential for DNA replication, binding nascent 1L.W.G. and K.R.L. contributed equally to this work. ssDNA that is generated when duplex DNA is unwound and 2Present address: Department of Biochemistry and Molecular Biology, Colorado State thereby preventing reannealing and/or the formation of secondary University, Fort Collins, CO 80523. structures that would impede progression of the replisome. Both 3Present address: Stem Cell Program and Divisions of Hematology/Oncology and Pulmo- proteins are also central to the cellular response to DNA lesions. nary & Respiratory Diseases, Boston Children’s Hospital, Boston, MA 02115. Although RPA and SSB exhibit no sequence homology, they each 4Present address: Department of Chemistry, Xavier University of Louisiana, New Orleans, employ an array of OB-folds for contacting ssDNA. Detailed LA 70125. analysis of RPA has revealed that it utilizes these OB-folds to 5To whom correspondence should be addressed. Email: [email protected]. contact ssDNA in distinct modes, engaging differing lengths of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ssDNA with different subunits, presumably to orchestrate higher- 1073/pnas.1722147115/-/DCSupplemental. order manipulations (6–8). Thus, to interact consistently throughout www.pnas.org/cgi/doi/10.1073/pnas.1722147115 PNAS Latest Articles | 1of6 Downloaded by guest on October 1, 2021 affinity is achieved through multivalency (2, 4, 8, 12–15). However, effects on specificity in vitro. These specificity mutations reduced ssDNA binding by the telomere-dedicated RPA complex (t-RPA) the ability of Cdc13 to tolerate variations in telomere sequence, is notably distinct from RPA, suggesting that these structurally which substantially impaired Cdc13 function in vivo. Moreover, similar domains have taken on distinct biochemical roles. Unlike the magnitude of the in vivo defect closely correlated with the RPA70, which uses two OB-fold domains for its core recognition of extent to which specificity was altered, thereby demonstrating ssDNA, Cdc13 employs a single OB-fold augmented by an un- that both affinity and sequence tolerance contribute to biological usually long β2–3 loop (Fig. 1) (16) to contact DNA with excep- function. Thus, by conducting a systematic analysis of the Cdc13 tionally tight picomolar affinity. Furthermore, Cdc13 binds ssDNA DNA-binding domain (DBD) interface, we have uncovered a with exquisite specificity for G-rich sequences (17, 18), which it finely tuned binding rheostat of specificity and affinity that achieves through recognition of a GxGT motif embedded in a confers optimal biological function. larger oligonucleotide (10, 12). Nevertheless, Cdc13 presumably needs to show sequence flexibility to accommodate the heteroge- Results neity of yeast telomeres (19), although the mechanism by which Systematic Mutagenesis of the DNA-Binding Interface of Cdc13 Cdc13 achieves this flexibility has not previously been elucidated. Identifies a 35-Fold Span in Affinity. To address how the bio- The sequence specificity and affinity displayed by Cdc13 pro- chemical features of Cdc13 allow it to perform its biological vide a unique system for investigating how these two biochemical roles, we introduced a set of eight alanine mutations into the properties contribute to function in vivo. To do so, this study DBD across the binding interface (16, 20), with an emphasis on examined an extensive panel of mutations across the DNA- the aromatic residues that play key roles in affinity and speci- binding interface for their effects on both binding affinity and ficity, and measured the impact of these changes on binding specificity and subsequently determined how perturbations in characteristics (Fig. 1A). The change in binding affinity to the either property affected Cdc13 function in vivo. Not