Understanding Single-Stranded Telomere End Binding by an Essential Protein
Final rept. 1 Aug 1999-31 Jul 2002
COLORADO UNIV AT BOULDER
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Cdcl3p is an essential protein from S. cerevisiae that binds to the single-stranded ends of telomeres with high specificity and affinity. Cdcl3p perform functions in concert with two protein complexes - protecting the end of the chromosome from degradation and regulating telomere length through the enzyme telomerase. Cdcl3p binds yeast single-stranded telomeric DNA sstelo DNA in vitro with high affinity Ksub d0.3 nM. The modular DNA-binding domain of the protein has been mapped by deletion analysis and proteolysis. We are investigating the structural and biochemical basis for high affinity binding and sequence specificity of the single-stranded DNA binding domain. We have used heteronuclear, multidimensional NMR spectroscopy to solve the high-resolution solution structure of the domain bound to a single-stranded telomeric DNA 11-mer. We have used isotope filtered and selectfilter NOE experiments to determine the single-stranded DNA conformation in the complex and observe protein-DNA contacts. Also, the thermodynamic contribution of amino acids at the interface has been probed by site-directed mutagenesis and filter-binding.
- Anatomy and Physiology
- Medicine and Medical Research