Telomeres, the ends of chromosomes, are essential for the stable maintenance of linear DNA molecules. Cytological studies suggest that telomeres are involved in the organization, pairing, segregation, and movement of chromosomes during mitosis and meiosis. While naked DNA and broken chromosomes are subject to extensive nuclease degradation and high levels of recombination, chromosomes with intact telomeres are stably maintained. Telomeres must be replicated and maintained in the cell by a mechanism that ensures the complete synthesis of both daughter DNA strands. Recent molecular studies suggest that telomeres may be replicated and/or maintained via a template- independent addition activity. Telomeric DNA probably interacts with specific enzymes and structural proteins to facilitate the complete and accurate replication, segregation, and maintenance of linear chromosomes. Our long term goal is to understand, at the molecular level, the mechanisms of these telomere functions, using the yeast, S. cerevisiae, as a model system. A telomere-binding activity from yeast binds specifically to sequences within the poly (C1-3A) tracts-that are found at and near the extreme termini of yeast chromosomes. In this project we propose to examine the characteristics of this telomere- binding activity. Different approaches will be used including: purification of the protein by column chromatography including DNA affinity chromatography; determination of the exact sequence an/or structural characteristics of the binding substrate by deletion analysis, DNase I protection and alkylation interference experiments; cloning of the gene or genes that encode this activity using anti-bodies and expression libraries; characterization of these genes, the regulation of their expression and the phenotype of null mutants generated by gene replacement experiments; and localization of the activity in vivo by cross- linking experiments.
| Petracek, M E; Lefebvre, P A; Silflow, C D et al. (1990) Chlamydomonas telomere sequences are A+T-rich but contain three consecutive G-C base pairs. Proc Natl Acad Sci U S A 87:8222-6 |
