Field of study: Biology
Lab: Diego Loayza
School: Manhattan Hunter Science High School
Class: 10th/ 11th Grade
For this 2008 summer internship, I have worked with Dr. Diego Loayza in his lab. We studied telomere replication in human cells. Telomere is an important parameter in cancer. They are made up of the sequence TTAGGG repeated thousands of time. Telomeres end with a single stranded overhang. And to that single stranded overhang, there is an enzyme called telomerase that will synthesize the TTAGGG repeat sequences. Telomere is also made up of specific proteins that bind to the repeat sequences TTAGGG and this complex is called shelterin.
The goal of our lab was to study C-stranded synthesis because it is required for telomere maintenance and elongation, in addition to the necessary action of telomerase. To achieve this goal, we started the analysis of known DNA replication components at telomeres. We focused on FEN1 and POLA2 because they are known to be essential for C-strand synthesis in yeast.
We asked whether FEN1 or POLA2 were associated with specific shelterin components at telomeres. To this end, we did a co-immunoprecipitation experiment. We detected FEN1 in a complex with POT1 in HTC75 cells, in a MYC-FEN1 overexpressing cell line. A negative control included a non-overexpressing cell line (vector only). This experiment shows that FEN1 and POT1 are together in a complex in HTC75 cells.
We also performed chromatin immunoprecipitations (ChIP). The result of this experiment was that FEN1, TRF1, POT1 and possibly POLA2 were detected. This was done by using the probe TTAGGG to detected telomeric sequences. An Alu repeat probe was used as a negative control for internal chromosomal sequences.
For future experiments, we plan on studying the modalities of the FEN1-shelterin interaction, and the significance of this interaction in telomere function.