Stimulation of de novo telomere addition by the Rap1 ... · Stimulation of de novo telomere...

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’

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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

t 1

kb la

dd

er

Λ

+φx

DN

A m

arke

r H

ot

1kb

lad

de

r H

ot

1kb

lad

de

r G

BD

+Rap

1C

#6

W

ild-T

ype

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