Dataset Information

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' 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.

SUBMITTER: Croy JE

PROVIDER: S-EPMC3987967 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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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 Johnny E JE Fast Jonas L JL Grimm Nicole E NE Wuttke Deborah S DS

Biochemistry 20080321 15

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 ...[more]

PMID: 18355038

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Project description:In the fission yeast Schizosaccharomyces pombe, the transcriptional-regulatory network that governs flocculation remains poorly understood. Here, we systematically screened an array of transcription factor deletion and overexpression strains for flocculation and performed microarray expression profiling and ChIP-chip analysis to identify the flocculin target genes. We identified five transcription factors that displayed novel roles in the activation or inhibition of flocculation (Rfl1, Adn2, Adn3, Sre2, and Yox1), in addition to the previously-known Mbx2, Cbf11, and Cbf12 regulators. Overexpression of mbx2(+) and deletion of rfl1(+) resulted in strong flocculation and transcriptional upregulation of gsf2(+)/pfl1(+) and several other putative flocculin genes (pfl2(+)-pfl9(+)). Overexpression of the pfl(+) genes singly was sufficient to trigger flocculation, and enhanced flocculation was observed in several combinations of double pfl(+) overexpression. Among the pfl1(+) genes, only loss of gsf2(+) abrogated the flocculent phenotype of all the transcription factor mutants and prevented flocculation when cells were grown in inducing medium containing glycerol and ethanol as the carbon source, thereby indicating that Gsf2 is the dominant flocculin. In contrast, the mild flocculation of adn2(+) or adn3(+) overexpression was likely mediated by the transcriptional activation of cell wall-remodeling genes including gas2(+), psu1(+), and SPAC4H3.03c. We also discovered that Mbx2 and Cbf12 displayed transcriptional autoregulation, and Rfl1 repressed gsf2(+) expression in an inhibitory feed-forward loop involving mbx2(+). These results reveal that flocculation in S. pombe is regulated by a complex network of multiple transcription factors and target genes encoding flocculins and cell wall-remodeling enzymes. Moreover, comparisons between the flocculation transcriptional-regulatory networks of Saccharomyces cerevisiae and S. pombe indicate substantial rewiring of transcription factors and cis-regulatory sequences.

Dataset Information

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

Publications

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

Similar Datasets

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