Another idea

Most of my research as an undergraduate focused on telomeric length regulation. However, one of the limitations of the lab that I was working in at the time was that, due to financial constraints, we were not able to do protein work. One aspect that particularly interested me was better understanding the interactions between telomerase and the various proteins associated with the telomere.

The yeast K. lactis contains 12 telomeres that are composed of 10-20 repeats of a uniform 25 base pair sequence that is unique to the telomeres. The major telomeric length regulation pathway is known to be mediated by telomerase, which reverse transcribes telomeric repeats from its RNA template and concatenates them onto the end of the telomere. A major question regarding telomerase activity is what signals telomerase to replenish the telomeric tip. Telomere binding proteins have been shown to be crucial toward both recruiting telomerase to the end and inhibiting its access to the DNA.

One yeast telomeric protein clearly associated with the length-regulation mechanism is the Rap1 telomeric DNA-binding protein. Previous work indicates that telomere length in yeast is regulated through a pathway involving Rap1. Rap1 binds double-stranded sequence along the length of the telomere. Counting of Rap1 molecules at telomeres has been shown to be crucial to telomere length regulation. Each telomeric repeat of a variety of yeast species contains a Rap1 binding site. Telomere mutations disrupting Rap1 binding lead to greatly increased telomere length. The left portion of the K. lactis telomeric repeat is thought to have a negative regulatory function. Rif1 and Rif2 are accessory proteins that interact with the C-terminus of Rap1 and have also been shown to negatively regulate telomere length.

A variety of other proteins contribute to telomere maintenance in yeast. Cdc13 is a single-strand telomere binding protein that recruits or activates telomerase and also appears involved in negatively regulating telomerase. The MRX complex has been shown to be required for telomerase-mediated telomeric addition, probably by helping recruit telomerase to the telomeres. Two DNA damage response kinases, Tel1 and Mec1, also contribute to telomerase's ability to add new sequence onto telomeric ends. It has been shown that the Rap1-based repeat counting mechanism is at least partially dependent on Tel1 expression, but the nature of this epistatic dependence is not well characterized.

The goal here is to dissect the interaction between Tel1 and the Rap1/Rif telomerase regulation pathway. Some basic ideas I had on how I could do this:

- use FRET to observe the dynamics between the different components of this pathway
- attempt to crystallize components of this pathway, then try to dissect the pathway by computation (probably not practical, since there's quite a few relevant proteins, and Rap1 may have been crystallized, but I don't think any of the others have)
- generate multiple knockouts to figure out how many independent control points are involved in this pathway (e.g. cdc13/tel1 double deletion, what happens to the elongation phenotype?)
- use microarrays to determine which genes are being transcribed as the telomeres shorten
- use microarrays to determine how gene expressions differ between WT and mutants with runaway telomere elongation