(Developmental Center-Driven Research Project)
UCSF: E Blackburn, D Levy, T Williams,
LLNL: R Balhorn, L Brewer,
UC Davis: V Heinrich, S Fore, P Yu-Yang, H Wu, Y Yeh
The number of times that a given eukaryotic cell line can divide and replicate is determined by the length of the telomere that protects the ends of each chromosome. Under normal circumstances, the telomere (which consists of tandem repeats of thymine and guanine: TGTGG) is incrementally shortened after each cell division. Telomerase is an enzyme that is able to repair telomeres (and potentially allow healthy cells to live forever), but its activity is regulated by a complex of DNA-binding proteins known as the telomere endcap. While most of the proteins associated with the endcap have been identified – including the repressor activator protein Rap1, and Rap1-interacting factors, Rif1 and Rif2 – the nature of their interaction with telomeric DNA is generally not well understood. We have developed methods to probe the detailed mechanisms of this complex regulatory machinery at the single molecule level. Techniques being used include atomic force microscopy (AFM), dual optical trapping and reflective interference trapping force microscopies, FRET - FCS, and antibunching spectroscopy. A more complete understanding of these interactions will lead to potential new medical solutions against diseases related to premature aging (shortened telomere) or cancer (cell immortality).
We have made progress on developing a novel DNA-chip for analyzing molecular mechanisms of DNA binding proteins using single molecule biophotonic techniques. Differences between organisms such as yeast and humans vary slightly and overall follow a similar structure. In the yeast organism, S. cerevisiae, the repeats are typically no longer than 18 base pairs and consist of two regions within the repeat. By constructing synthetic oligonucleotides containing this sequence, we will look at structural and kinetic aspects of Rap1 binding to this sequence.
We have developed a functionalized quartz coverslip (chip) that allows easy attachment of double stranded telomeric DNA probes to its surface in order to understand how Rap1 bends telomeric DNA so as to initiate the formation of the endcap complex. This has been accomplished by determining the strength and kinetics of Rap1 interaction with DNA using optical force microscopy and pulsed-interleaf-excitation fluorescence-resonance-energy-transfer (PIE)-FRET combined with fluorescence correlation spectroscopy (FCS). In addition reflective interference optical trapping microscopy has been used to measure changes in elasticity and tension of the binding event employing reflective interference optical trapping microscopy. In summary, this year we have been able to use fluorescent labels to measure distances (with PIE-FRET) and have shown in model systems (protamine/spermine) that the DNA strands are shortened with protein attachment.