Shyam Murali Mentor: Dr. Katherine Friedman
Stimulation of de novo telomere addition by the Rap1 protein of
yeast
CEN
W W W
Correct repair Incorrect repair GCR
Telomere addition
Translocation/deletion Resume cell cycle
Sequence loss
“Damage tolerance” De novo
Fates of a double-stranded break
Image from Dr. Friedman
No repair Resection
Cell death
Telomeres
• Repetitive sequence at the end of chromosome
• Protect ends of chromosome from “end-replication” problem and chromosome fusion
• TG- and Protein-rich
• Telomerase
Telomere Hotspot
• Chromosome V – Saccharomyces cerevisiae
8.4 kb 83 bp Highly TG-rich Rap1 binding site
Essential WWW Hotspot HO HYGR URA3
3 kb
Image from Kati Turner
HO Endonuclease Assay
8.4 kb 83 bp Highly TG-rich Rap1 binding site
Essential WWW Hotspot HO HYGR URA3
3 kb
GAL10 HO
Image from Kati Turner
Multiplex PCR Centromere proximal Telomere proximal
WWW
PCM1 URA3 HS
*
HO HYGR
Hotspot
Image from Kati Turner
Telomere Hotspot
• Chromosome V – Saccharomyces cerevisiae
5’ –GGATGTAGGATGAGTTGGTGTGGTGTTACTACTAGGATTTGGCGTGGATGAAGGACCTGCAGTGGAGGGTGTTGTTGTGGAGTT- 3’
AAAAAAAAAAAAAAAAAA
Essential WWW HS HO
HYGR URA3
Rap1 Binding Site
PolyA Mutation
Image from Kati Turner
Preliminary HO Endonuclease Assay Data
Rif1 Rif2 TLC1
Est2
Rap1
The Rap1/Rif1/Rif2 complex has different effects
TLC1
Est2 +
Centromere Telomere HOTSPOT
Rap1 Rap1 Rap1 Rap1
Rif1 Rif2
Rap1
ENDOGENOUS TELOMERE
Image from Dr. Friedman
Gal4 UAS Project: Important Objectives
• Is Rap1p sufficient for telomere addition at the hotspot?
• Determine which proteins, or protein domains, are required for de novo telomere addition
• Hypothesis:
• Rap1p is sufficient for telomere addition
• Rap1p C-terminus is sufficient
How will we do this?
UAS Sequence creation and integration
• Created a new strain
– JRL017 91-2 Rap1BS [UAS] Hxt13::URA3
• Used PCR to create constructs and cloned into pRS306
Rap1 Binding site UAS Mutation
TG rich (telomere-like)
HindIII
XbaI
XbaI
HindIII
Image from Jacob Seloff
Two-Step Integration
Plated On 5-FOA
URA3
1.
2.
BamH1 Cut Site
URA3
-Mutated Hotspot
-Deleted Hotspot
URA3
Image from Jacob Seloff
UAS strain Gal Assay
• Expectation: replacement of Rap1p binding domain should yield results similar to Rap1BS PolyA Mutant
Integration of Gal4 DBD constructs
1. Gal4 DBD (alone)
2. GBD+Rap1 fusion
3. GBD+Rap1c fusion
Only C-terminus of Rap1
How?
1. Clone GBD constructs into pRS304 (integrative) and pRS314 (centromeric)
2. Transform into UAS strain of yeast
3. Confirm Integration
4. Perform HO Endonuclease Assay
Southern #1 – GBD Probe
Southern #2 – TRP1 Probe
Ho
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lad
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GBD only GBD+Rap1
Future Goals
• Western Blot
• If GBD constructs have correct expression HO Endonuclease Assay
• Disparity between Positive (at hotspot) vs. Negative (at telomeres) regulation of telomerase by Rap1p. Why?
• Does number matter?
• Multiple UAS sites for more Rap1 recruitment
• Rif1p/Rif2p fusion?
Does the number of Rap1 molecules matter?
Caveat - Directionality
• Directionality of Rap1p
• Use other non-hotspot Rap1p binding sites
Acknowledgements
• Lab Members:
• Dr. Katherine Friedman
• Margaret Platts
• Udo Obodo
• Ann Ding
• Kati Turner
• Laura Bechard
• Charlene Hawkins
• Committee Members:
• Dr. Woelfle
• Dr. Rokas
• Honors Program Coordinator
• Dr. Patton
• Beckman Scholars Program
• Dr. Johnston