Understanding Single-Stranded Telomere End Binding by an Essential Protein
Abstract
Telomeres are the nucleoprotein structures that cap the ends of eukaryotic chromosomes. Telomere length 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. Cdc13p is an essential protein from S. cerevisiae that binds to the single-stranded ends of telomeres with high specificity and affinity. Genetically, Cdc13p has been shown to protect the end of the chromosome from degradation and to load telomerase in concert with the protein Est1p. Biochemically, Cdc13p binds yeast single-stranded telomeric DNA (sstelo DNA) in vitro with high affinity (K(sub d) = 0.3 nM). The DNA-binding domain of the protein has been mapped previously. We are investigating the structural and biochemical basis for high affinity binding and sequence specificity of this domain. A high resolution solution structure of the protein/DNA complex is in progress. Here we use NNR experiments to determine the single-stranded DNA conformation in the complex and directly observe protein/DNA NOE contacts. In a complementary approach, we have performed in vitro protein/DNA photocrosslinking experiments using the chromophore 5-iodouracil. Proteolytic digestion and peptide micro-sequencing have allowed identification of sites in the protein involved in binding ssDNA.
Document Details
- Document Type
- Technical Report
- Publication Date
- Aug 01, 2001
- Accession Number
- ADB282191
Entities
People
- Emily Anderson
Organizations
- University of Colorado Boulder