Effect of Cancer-Associated Mutations on MLH1 Interaction

with Exonuclease

Gautam MankaneyGautam Mankaney

Mentor: Dr. Andrew BuermeyerMentor: Dr. Andrew Buermeyer

Howard Hughes Medical InternshipHoward Hughes Medical Internship

CANCER

Cancer

• Uncontrolled proliferation

of cells

• 2nd leading cause of death

(500 thousand/ yr)

Causes

• Cellular mutations

• Inherited or Sporadic

Colorectal Cancer• 3rd most common

cancer (10.7%)• $8.4 billion in

treatment costs• 3-5% cases linked

to Lynch Syndrome

(HNPCC)

New Cancer Incidents by State

Detected MMR Genes in Lynch Syndrome Families

Lynch Syndrome (HNPCC)

•80% develop colorectal cancer

other cancers include: kidney, stomach,

ovary, small bowel, pancreas , & bile duct •Mismatch Repair Deficiency

0

10

20

30

40

50

60

70

80

90

100

MLH1/ MSH2 MSH6 PMS2

Detected Mutations (%)

MMR Genes

DNA Repair Mechanisms - Mismatch Repair (MMR)

DNA Damage

•Environmental (carcinogens, UV light)

•Metabolic activities (free radicals, replication)

DNA Mismatch Repair

•Evolutionarily conserved process

•Fidelity of DNA replication

a) Base substitution, insertion, and

deletion mismatches and loops

b) DNA lesions - environmental and

intracellular stress

c) Apoptosis

•MMR loss - multistage carcinogenesis

PROKARYOTIC MMR

CH3 CH3

mismatch

MutS, MutL, MutH

CH3 CH3

mismatch

CH3 CH3

mismatch

5’

5’

5’

5’

5’5’

3’

3’

3’

3’3’

3’

5’ nick 3’ nick

Exonuclease - ExoVII or RecJ Exonuclease - ExoIHelicaseII HelicaseII

CH3 CH3

5’

5’

3’

3’CH3 CH3

5’5’3’3’

DNA Polymerase IIIDNA Ligase DNA Ligase

DNA Polymerase III

CH3 CH3

5’

5’

3’

3’

MutLα – Understanding the Structure

•MLH1 – PMS2

ExoI

3 241 756492 711621

Dimer interface

ssDNA binding

ATP-binding/ hydrolysis

PMS2, EXO1interaction

C-terminalhomology

Linker

MLH1 - functional domains

Research QuestionResearch Question Compared to MLH1 wildtype and and non-

pathogenic polymorphisms, how well does ExoI interact with certain MLH1 mutants?

- L582V, K751R, L607H, and R755W

-putatively associated with Lynch Syndrome

-do not affect MLH1 protein stability

-do not affect MLH1 - PMS2 interaction

3 241 756492 711621

Dimer interface

ssDNA binding

ATP-binding/ hydrolysis

PMS2, EXO1interaction

C-terminalhomology

Linker

Hypothesis Compared to MLH1 wildtype and MLH1

non-pathogenic polymorphisms, L582V, K751R,

L607H, and R755W will show a decreased ability in binding

EXO1

- in vitro assays that measure interaction capabilities

MLH1 - functional domains

ApproachApproach1.Construct in vitro expression vectors containing coding

regions to be expressed

- MLH1 Wildtype

- MLH1 Mutants: L582V, L607H, K751R, R755W

- ExoI

2. Find a way to probe for ExoI

-express protein and test antibody

3. Perform in vitro co-immunoprecipitation assays

with ExoI, PMS2, and MLH1 variants

Plasmid ConstructionPlasmid Construction

•Excision of cDNA by restriction digestion

•Gel isolation of cDNA

hMLH1

wt

•Ligation into linear pCite vector

pCite

•Restriction digests (screening)

and sequencing

pCMV

pCMV

hMLH1 wt

hMLH1 mutant

hMLH1

mutant

hMLH1hMLH1

mutant

Constructing the PlasmidConstructing the Plasmid

(Three Way Ligation)(Three Way Ligation)Isolate Fragments

MLH1(part) MLH1(part containing mutation)

hMLH1

wthMLH1

mutantpCite

hMLH1hMLH1

mutant

Plasmid Screening

pCite

hMLH1hMLH1

mutant

restriction enzyme (Xho1)

hMLH1

mutanthMLH1

2.4 kb

3.8 kb

Restriction Digests

Qu

ickTim

e™

an

d a

TIF

F (U

nco

mp

res se

d) d

eco

mp

ress

or

are

need

ed

to se

e th

is pic

ture

.

ladder 1 2 3* 4 5 6* 7 8 9 10 11 12 13 14*

2000 kb2500 kb

3000 kb4000 kb5000 kb6000 kb

10,000 kb

Qu

ickTim

e™

an

d a

TIF

F (U

nco

mp

resse

d) d

eco

mp

resso

rare

need

ed

to se

e th

is pic

ture

.

15 16 17* 18* 19 20 21* 22* 23* 24ladder

2000 kb

10,000 kb

2500 kb3000 kb

4000 kb5000 kb6000 kb

K751W R755W

L582V L607H

Co-immunoprecipitaton

Transcription

Translation

hMLH1

pCite

hEXO1PMS2

pCITE

Antibody – PMS2

Antibody Binding Beads

Co-immunoprecipitatio

n

WashWestern Blot

pCITE

Example Co-immunoprecipitation

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.•Qualitative Measurement

Exonuclease DetectionhEXO1

pCITE

Transcription

Translation

protein

Exonuclease ≈ 105 kD

MCF-7 30ul 15ul 10ul 5ul MCF-7 30ul 15ul 10ul 5ul

250

150

100

75

50

U.S Biological

(mouse monoclonal Ab)

NeoMarkers

(mouse monoclonal Ab)

Questions

1. Is protein being produced?

2. Is there a problem with the detection method?

Flagging hEXO1Flagging hEXO1

•Octapeptide - DYKDDDDK

•Polymerase Chain Reaction (PCR)

hEXO1hEXO1

Flag-Exonuclease Detection

250

150

100

75

50

Dr. Binghui Shen

City of Hope National Medical Center

and Beckman Research Institute

Flag-Exonuclease30ul 15ul 10ul 5ulExo 30ul 15ul 10ulExo

15mg protein

4.5ng 2.2ng 1.5ng .75ng 1.5ng

rabbit polyclonal

anti-Flag

Anti-Flag

4.5ng 2.2ng 1.5ng 1.5ng

Summary

Insufficient antibody sensitivity to ExoI with

two different mouse monoclonal Ab’s

Constructed prokaryotic transcription vectors

For MLH1 WT, MLH1 mutants, ExoI

Added flag peptide to amino terminal

of ExoI reading frame

Insufficient antibody sensitivity to ExoI with

Anti-Flag

Future StudiesFuture Studies1.FLAG carboxyl end of hEXO1

2.Try a different epitope tag

3.35S labeled Methionine

1Schmutte, C., M. M. Sadoff, S. Guerrette, S. Acharya, and R. Fishel. Interactions of the human exonuclease I with DNA mismatch repair proteins hMSH2, hMSH3 and hMLH1. J. Biol. Chem., in press.

2 Tran, P. T., J. A. Simon, and R. M. Liskay. Interaction of EXO1 with components of MutLα in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA, in press.

4. 1Glutathione-S-transferase protein-protein interaction assay

-GST fusion protein (PMS2)

-35S labeled ExoI

5. 2Two Hybrid Assay

- PMS2 fused to a binding domain

- ExoI fused to activation domain

Thank You!Thank You!

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

•The Buermeyer Lab

Dr. Andrew Buermeyer

Dr. Scott Nelson

•Howard Hughes Medical Institute

•Undergraduate Research, Innovation, Scholarship & Creativity (URISC)

•Dr. Kevin Ahern

CTD

NTD

CTD

NTD

Exonuclease

DNA Mismatch Repair (MMR)

2) The PMS2 mutants will show a decreased ability to bind

MLH1 compared to PMS wildtype

2) The PMS2 mutants will show a decreased ability to bind

MLH1 compared to PMS wildtype

Perform LigationsUsing T4 DNA Ligase

Schmutte, C., M. M. Sadoff, S. Guerrette, S. Acharya, and R. Fishel. Interactions of the human exonuclease I with DNA mismatch repair proteins hMSH2, hMSH3 and

hMLH1. J. Biol. Chem., in press.

74a.Tran, P. T., J. A. Simon, and R. M. Liskay. Interaction of EXO1 with components of MutLα

in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA, in press.