Examining the Mechanism of Telomere Overlongation by est2 Mutants in S' cerevisiae

1
Examining the Mechanism of Telomere Overlongation
by est2 Mutants in S. cerevisiae
Chris AdkinsDr. Katherine FriedmanBSCI 296
2
The Telomerase Complex
3
Processivity of Telomerase
4
Long-telomere mutants of Est2p



Ji, Adkins, et al., 2008
5
Telomere Sequences Are Altered in est2 Strains
GG dinucleotide incorporation
P 0.012
Ji, Adkins, et al., 2008
6
Measuring Telomerase Processivity
GA426 est2?TRP pKF410-proA-EST2 or
-proA-est2-LTE76K or -proA-est2-up34
Make extract
ProA IP
Telomerase Assay
7
In Vitro Primer Extension
Est2p
484
468
3
5
TLC1 RNA
ACACACACCCACACCAC
Tel14
GTGTGGTGTGGG
5
a-32PdGTP
8
In Vitro Primer Extension
Est2p
484
468
3
5
TLC1 RNA
ACACACACCCACACCAC
1
Tel14
GTGTGGTGTGGGT
5
a-32PdGTP
9
In Vitro Primer Extension
Est2p
484
468
3
5
TLC1 RNA
ACACACACCCACACCAC
1 2
Tel14
GTGTGGTGTGGGTG
5

a-32PdGTP
10
In Vitro Primer Extension
Est2p
484
468
3
5
TLC1 RNA
ACACACACCCACACCAC
1 2 3
Tel14
GTGTGGTGTGGGTGT
5

a-32PdGTP
11
In Vitro Primer Extension
Est2p
484
468
3
5
TLC1 RNA
ACACACACCCACACCAC
1 2 3 4
Tel14
GTGTGGTGTGGGTGTG
5


a-32PdGTP
12
In Vitro Primer Extension
Est2p
484
468
3
5
TLC1 RNA
ACACACACCCACACCAC
1 2 3 4 5
Tel14
GTGTGGTGTGGGTGTGG
5



a-32PdGTP
13
In Vitro Primer Extension
Est2p
484
468
3
5
TLC1 RNA
ACACACACCCACACCAC
1 2 3 4 5 6
Tel14
GTGTGGTGTGGGTGTGGT
5



a-32PdGTP
14
In Vitro Primer Extension
Est2p
484
468
3
5
TLC1 RNA
ACACACACCCACACCAC
1 2 3 4 5 6 7
Tel14
GTGTGGTGTGGGTGTGGTG
5




a-32PdGTP
15
In Vitro Primer Extension
Examining NAP
a-32PdGTP
16
In Vitro Primer Extension
est2-up34 Telomerase Exhibits Decreased NAP in
vitro
1 2 3 4 5 6 7
1 2 3 4 5 6 7
P values calculated by ANOVA
17
In Vitro Primer Extension
Primer Mutants Permit RAP in vitro
Tel14 GTGTGGTGTGTGGG3 Tel15(m4,5)
TGTGTGGTGTCAGGG3
Adapted from Bosoy and Lue, 2004
18
In Vitro Primer Extension
Lue and Li Report that E76K Exhibits Increased
RAP in vitro
Tel14 GTGTGGTGTGTGGG3 Tel15(m4,5)
TGTGTGGTGTCAGGG3 OXYT1 GTTTTGGGGTTTTGGG-3
Lue and Li, 2007
19
In Vitro Primer Extension
Est2-LTE76Kp
Est2-LTE76Kp
Est2-LTE76Kp
Est2-LTE76Kp
Est2-up34p
Est2-up34p
Est2-up34p
Est2-up34p
Est2p
Est2p
Est2p
Est2p
1 2 3 4 5 6
7 8 9 10 11 12
13 14 15 16 17 18
19 20 21 22 23 24
gt First Round
gt First Round
First Round
First Round
Short Exposure
Long Exposure
Short Exposure
Long Exposure
20
In Vitro Primer Extension
I Report That Est2-LTE76Kp Does Not Exhibit
Increased RAP and that Est2-up34p Exhibits
Decreased RAP
-4.7
-40
P 0.646
P 0.005
n 4
P values calculated by ANOVA
21
In Vitro Primer Extension
I Report That Est2-LTE76Kp Does Not Exhibit
Increased RAP and that Est2-up34p Exhibits
Decreased RAP
-0.6
-10
P 0.887
P 0.021
n 4
P values calculated by ANOVA
22
Conclusions and Future Work

• Altered processivity does not contribute to
  increased GG dinucleotide incorporation
• Est2-LTE76Kp does not exhibit changes in NAP or
  RAP
• Est2-up34p exhibits decreases in both NAP and RAP
• Examine other enzymatic properties such as TLC1
  pairing with primer and substrate binding

23
Acknowledgements

• Dr. Katherine Friedman
• Dr. Hong Ji
• Bethany Cartwright
• Other lab members
• The Lue and Zakian labs

24
Assay Concentrations
25
Rap1p a negative regulator of telomere length
Rap1p
Not elongated
Subtelomeric region
Telomeres
Elongated
26
The frequency of Rap1p binding is decreased in an
est2-LT background
Ji, Adkins, et al., 2008
EST2
Subtelomeric region
Telomeres
Rap1p
est2-LTE76K
Subtelomeric region
Telomeres
est2-up34