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Deciphering the mechanism of thermodynamic accommodation of telomeric oligonucleotide sequences by the Schizosaccharomyces pombe protection of telomeres 1 (Pot1pN) protein.

Croy JE, Fast JL, Grimm NE, Wuttke DS

Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.

Linear chromosomes terminate in specialized nucleoprotein structures called telomeres, which are required for genomic stability and cellular proliferation. Telomeres end in an unusual 3' single-strand 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.

Published 9 April 2008 in Biochemistry, 47(15): 4345-58.
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