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Understanding Single-Stranded Telomere End Binding by an Essential Protein

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Annual summary rept. 1Aug 1999-31 Jul 2000

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Telomeres are the nucleoprotein structures that protect the ends of eukaryotic chromosomes. Telomere length regulation is controlled by the enzyme telomerase and a suite of telomere binding proteins. Anomalous telomeric replication and regulation are implicated in most forms of cancer, while telomeric shortening contributes to cellular aging. Cdcl3p is an essential protein from S. cerevisiae that binds to the single-stranded ends of telomeres with high specificity and affinity. Genetically, Cdcl3p has been shown to protect the end of the chromosome from degradation and to load telomerase. Biochemically, Cdcl3p binds yeast single-stranded telomeric DNA sstelo DNA in vitro with high affinity KdO.3 nM We are investigating the structural basis for high affinity binding and sequence specificity of the single-stranded DNA binding domain. We have expressed and purified the ssDNA binding domain in high yield. Its binding affinity and specificity have been examined with libraries of sstelo DNA randomized at each position. In vitro photocrosslinking experiments have been performed using 5- iodouracil substituted for thymine bases. Proteolytic digestion of the crosslinked products along with micro peptide sequencing have allowed us to identify sites critical for DNA binding. These experiments complement high resolution NMR studies of the proteinDNA complex in progress in our laboratory.

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  • Biochemistry
  • Genetic Engineering and Molecular Biology
  • Anatomy and Physiology
  • Medicine and Medical Research

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