Lab Meeting 81314 Trisha Ablordeppey Telomeres A Telomerase

Lab Meeting 8/13/14 Trisha Ablordeppey

Telomeres A Telomerase 3’ B 5’ CCC U AA CST 3’ Slide By Jason Stewart

Functions of CST Fork stalling due to structure at difficult -toreplicate DNA CST Telomere Replication Tpp 1 Telomerase Pot 1 CST ? ? Polα/primase Slide By Jason Stewart

Project Overview • Knockdown of CST led to increase in MTS • Does this knockdown lead to more gaps/breaks at fragile sites?

Metaphase Spreads • Treatment of sh. STN 1, RES, NT He. La Long cells with. 25 ug/m. L Aphidicolin for 16 hours – Aphidicolin inhibits DNA polymerase • Cells were then treated with Colcemid, to stop cells in Metaphase stage • Cells were collected and fixed to slides, and stained with Giemsa sh. STN 1+APH

Results NT NT+APH Trial 1 2. 34% Trial 2 0% Trial 3 0% Trial 4 RES 0. 89% RES+APH sh. STN 1+APH 1. 23% 1. 36% 0. 83% 4. 14% 0% 2. 67% 0. 43% 2. 3% 0% 0. 36% 3. 8% 0% 0. 70422535% 0. 37313433% 0% Percentage of Gaps/Breaks 4. 5 4 3. 5 3 2. 5 Percentage 2 Trial 1 Trial 2 1. 5 Trial 3 1 Trial 4 0. 5 PH sh ST N 1 +A ST N 1 sh H AP S+ RE RE S PH +A NT NT 0 Cell Type

Conclusions • Treatment with APH didn’t give consistent enough results • Very hard to find spreads on APH treated slides • Contamination problems, Jason leaving, and starting a new project led to abandoning project

Shelterin and CST Interaction CST M. Rajavel, et al. , 2014

Project Overview • CST binds ss. DNA and binds to TPP 1 • Interaction between TPP 1 -CST for CST function at telomere? • TPP 1 - Dominant Negative – Unable to Bind to Shelterin Complex, Telomerase and POT 1 First 86 A. A deleted for Truncated D 166 R, E 168 R Telomerase A. A 250 -344 Deleted POT 1 W 527 R, A 528 K TIN 2 M. Rajavel, et al. , 2014

Cloning

Confirmation of Gene in MIEG 3 Plasmid 1. 5 kb Restriction Enzyme Digestion Colony Screening Top Row – Full Length Bottom Row – Truncated

Problems With Truncated TPP 1 • Picked new colonies for screening • Ligated at Lower Temperature • Used more Insert for Ligation • Used More RE as Sma 1 has low cutting efficiency Conclusion • Bacteria did not like the DNA • Went ahead with Full Length Colony Screening with Truncated TPP 1

Establishment of TPP 1_DN Stable Line • Virus was made in HEK cells • After 24 hours, He. La Long cells were transduced • 48 hours after transduction, cells were sorted • Wild Type Cells were used as a standard Wild Type TPP 1 -DN

Future Goals Now • Western Blot – Confirm the presence of TPP 1 -DN • IP – Check to see if TPP 1 -DN still interacts with CST Later • Test if TPP 1 -DN affects the binding of shelterin at telomeres indirect immunofluorescence microscopy • Test if TPP 1 -DN prevents CST from binding to telomeres by Ch. IP • Test if TPP 1 -DN causes changes in telomere structure with FISH