Deciphering the Mechanism of Thermodynamic Accommodation of Telomeric Oligonucleotide Sequences by the Schizosaccharomyces pombe Protection of Telomeres 1 (Pot1pN) Protein

Abstract

Linear chromosomes terminate in specialized nucleoprotein structures called telomeres, which are required for genomic stability and cellular proliferation. Telomeres end in an unusual 3′ singlestrand overhang that requires a special capping mechanism to prevent inappropriate recognition by the DNA damage machinery. In Schizosaccharomyces pombe, this protective function is mediated by the Pot1 protein, which binds specifically and with high affinity to telomeric ssDNA. We have characterized the thermodynamics and accommodation of both cognate and noncognate telomeric single-stranded DNA (ssDNA) sequences by Pot1pN, an autonomous ssDNA-binding domain (residues 1-187) found in full-length S. pombe Pot1. Direct calorimetric measurements of cognate telomeric ssDNA binding to Pot1pN show favorable enthalpy, unfavorable entropy, and a negative heat-capacity change. Thermodynamic analysis of the binding of noncognate telomeric ssDNA to Pot1pN resulted in unexpected changes in free energy, enthalpy, and entropy. Chemical-shift perturbation and structural analysis of these bound noncognate sequences show that these thermodynamic changes result from the structural rearrangement of both Pot1pN and the bound oligonucleotide. These data suggest that the ssDNA-binding interface is highly dynamic and, in addition to the conformation observed in the crystal structure of the Pot1pN/d(GGTTAC) complex, capable of adopting alternative thermodynamically equivalent conformations.Protein recognition of nucleic acids is a key underlying molecular recognition event that guides replication, transcription, recombination, and other essential biological processes. To † We acknowledge the Wenner-Gren Foundation Postdoctoral Fellowship (to J.L.F.), National Research Service Award (NRSA) Postdoctoral Fellowship GM-071257 (to J.E.C.), and National Institutes of Health (NIH) GM-059414 (to D.S.W.), and National Science Foundation (NSF) (to D.S.W.) (MCB-0617956) for funding this research.© Copyright 2008 by the American Chemical Society * To whom correspondence should be addressed. date, a number of structural, biochemical, and thermodynamic studies have provided a fundamental understanding of how protein/double-stranded DNA (dsDNA) 1 recognition occurs. These studies reveal that dsDNA recognition is mediated by direct and indirect protein contacts that form intricate networks of hydrogen bonds with the bases (1-3). Thermodynamically, dsDNA recognition is generally characterized by negative enthalpy, positive entropy (ascribed to water/ion release), and large negative heat-capacity changes (ascribed to surface exclusion of the solvent) (4). Furthermore, the specific recognition of dsDNA typically leads to larger negative heat-capacity changes when compared to its nonspecific counterpart (1, 2). Although these characteristics are common, they can be modulated by many physical processes, including (1) conformational changes of the molecules upon binding (5), (2) redistribution of ion atmospheres, (3) dynamic behavior, and (4) hydration patte...

Structural anatomy of telomere OB proteins

“…At higher temperatures entropy opposed DNA binding and enthalpy compensated by becoming larger in magnitude. This same pattern of favorable enthalpy and unfavorable entropy terms was also measured for Sp -Pot1N binding with d(GGTTAC) and single-nucleotide substitution variants (Croy et al , 2008). These trends indicate that single-stranded telomere DNA binding is not strongly driven by the hydrophobic effect, consistent with few apolar atoms contributed by single-stranded DNA at the OB-DNA interface.…”

Telomerase and Telomere-Associated Proteins: Structural Insights into Mechanism and Evolution

“…These biochemical observations suggest that Sp Pot1 is capable of remarkable conformational plasticity and ligand accommodation. Supporting this hypothesis, structures of Sp Pot1pN complexed with non-cognate ligands identified novel ligand conformations that nonetheless bound with similar thermodynamic parameters (Croy et al, 2008). As the core S. pombe telomere sequence repeats are often separated by a variable number of nucleotides (Trujillo et al, 2005), the ability to accommodate alternate telomeric sequences with minimal thermodynamic impact may be an essential element of Sp Pot1 function.…”

“…4B). Solution dynamics analysis of the free and bound states of Sp Pot1pN suggests that specificity is achieved by a conformational selection mechanism where residues involved in forming the specific contacts experience dynamics that are quenched upon binding (Croy and Wuttke, 2009; Croy et al, 2008). …”

Insights into the Dynamics of Specific Telomeric Single-Stranded DNA Recognition by Pot1pN

“…Furthermore, we were able to assign Trp65, which was not visible in the 15 N HSQC spectrum of Pot1pN B due to exchange broadening (Supplemental Table 1). 24 The addition of Trp65 provides a complete residue assignment of the ssDNA-binding interface present in Pot1pN, as defined by the high-resolution crystal structure of Pot1pN B . 11 Finally, as observed in the assignment of Pot1pN B , the remaining unassigned residues in Pot1pN F cluster either to the N- and C-termini or to a region of the protein directly adjacent to the ssDNA-binding interface (Supplemental Table 1).…”