The goal of the proposal is to use mass spectrometric methods to identify and map protein interactions at telomeres in S. cerevisiae. Telomeres are dynamic DNA-protein complexes located at the termini of eukaryotic chromosomes maintaining a critical length of the telomere appears to be crucial for cellular viability.
Two specific aims are proposed for identification and mapping of telomeric proteins.
Specific aim 1 : Identification of protein-protein interactions at telomeres in S. cerevisiae. A genomic tag (IgG epitope from protein A) dll be placed on a known telomeric protein, and the proteins associated WITH the tagged protein will be isolated with IgG resin. Protein c complexes will be resolved by denaturing electrophoresis, and bands will be excised and subjected to proteolysis. Peptides and corresponding proteins will be identified by mass spectrometry and database searching.
Specific aim 2 : Napping protein interactions at telomeres in S. cerevisiae Protein complexes will be isolated as described in specific aim 1 and chemically cross-linked. Cross-linked proteins will be resolved by lD or 2D denaturing electrophoresis, and bands corresponding to cross-linked proteins will be excised and subjected to proteolysis. Mass spectrometry and database searching will be used to identify cross-linked proteins. By identifying the proteins containing the cross-linked peptides, information will be obtained on the spatial orientation of the telomeric proteins in the protein complex.
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