dna: mutation, repair and the...

DNA: Mutation, Repair and theEnvironment

Leona D. Samson

Biological Engineering Department, MIT

Biology Department, MIT

Center for Environmental Health Sciences, MIT

Center for Cancer Research, MIT

Computational and Systems Biology Initiative, MIT

Broad Institute, MIT

The Genetic Basis of Cancer andTheodor Boveri 1862 - 1915

• Established that chromosomescarry the hereditary information byshowing that aberrant segregationof chromosomes leads to certainphenotypes in sea urchin eggs.

• Suggested that aberrantsegregation of human chromosomescould be responsible for a normal cellbecoming a tumor cell

• Suggested that some chromosomespromoted cell growth and othersinhibit cell growthMarcella O’Grady Boveri (1865-1950) also contributed

Marcella O’GradyBoveri (1863-1950)also contributed to

Boveri’s theory

She was the firstwoman student to

graduate from MITwith a Biology Major

in 1885!

J Med Genet. 1985;22(6):431-40.Marcella O'Grady Boveri (1865-1950) and the chromosometheory of cancer

Chromosomesfrom a

Normal cell

Chromosomesfrom a

Tumor cell

Chromosomes from a PancreaticTumor Cell

http://iusd.k12.ca.us/uhs/cs2/images/DNA.jpg

2

Sunlight

Pollution &Food

Oxidation

NO.Cigarette Smoke

DNA is constantly beingdamaged by endogenous

and exogenous agents

Central Dogma of Molecular Biology

Damage to DNA cancreates permanent

changes in the geneticinformation

Inactive proteins orproteins with alteredfunction are produced

DISEASECell DeathMutation

DNA damage


DNArepair

DNA damage


DNArepair

DNA damage

Cellcyclearrest

Why do we care about DNA damagingagents in our environment??

Expo

sure

-

Expo

sure

-

Envi

ronm

ent

Envi

ronm

ent G

enetic

Genetic

Susceptibility

Susceptibility

Time/Age/BehaviorTime/Age/Behavior

HumanHealth/Disease

EatEat

BreatheBreathe InfectionInfection

AbsorbedAbsorbed

MedicineMedicine

EnvironmentalExposures

EnvironmentalExposures DrinkDrink

Food: www.boarhouse.ruDrink: www.terlyn.com

Air: www.npl.co.ukPharmaceutical: www.butterworth-

labs.co.ukSun: www.epa.gov

Helicobacter: microbewiki.kenyon.edu

One dramatic example -Xeroderma Pigmentosum

Lack of DNA Repair speeds up the carcinogenicprocess, presumably because mutations accumulate

more rapidly


DNArepair

DNA damage

Cellcyclearrest

Another dramatic example – deficiencies inDNA damage induced cell cycle arrest

THE FIRST SIGNS of ataxiatelangiectasia (A-T) usuallyappear in the second year oflife as a lack of balance and

slurred speech. It is aprogressive, degenerativedisease characterized bycerebellar degeneration,

immunodeficiency,radiosensitivity (sensitivity toradiant energy, such as x-ray)and a predisposition to cancer.

AtaxiaTelangiectasia

Ataxia Telangiectasia – Cancer Prone

Defective DNADamage Responses

can affect bothneurodegeneration

and cancersusceptibility

How do genes getmutated?

How can we stop themfrom being mutated?

EatEat

BreatheBreathe InfectionInfection

AbsorbedAbsorbed

MedicineMedicine

EnvironmentalExposures

EnvironmentalExposures DrinkDrink

Food: www.boarhouse.ruDrink: www.terlyn.com

Air: www.npl.co.ukPharmaceutical: www.butterworth-

labs.co.ukSun: www.epa.gov

Helicobacter: microbewiki.kenyon.edu

Contaminated Corn

ContaminatedPeanuts

Aspergillusflavus

Aflatoxin

Aflatoxin in food

HBV liver infection

Relative Risk

3 X

7 X

60 X

Exposures that increase risk ofHepatocellular carcinoma (HCC)

8,9-Dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1

N

NH

NH

N

NH2

O

OH

O

O

CH3

O

O

O

O

Normal Guanine

This Guanine has a large DNA adduct that is derivedfrom a compound secreted by mold that grows on

peanuts.

Normal DNA bases can become damaged when theyreact with chemicals that come from our food.

This is “junk” that doesnot belong on DNA.

G

AG

C

G

T

C

G

C

8,9-Dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1

N

NH

NH

N

NH2

O

OH

O

O

CH3

O

O

O

O

T

C

G

C

?

G

AG

C

G

T

C

G

C

A??

Original Sequence: CTCGC

Mutant Sequence: CTCGA

The ‘C’ was replaced with ‘A’

G

AG

C

G

How do genes getmutated?

How can we stop themfrom being mutated?

All these responses to DNA damage serve to preventmutations accumulating, and thus prevent CANCER

INSULT

RESPONSE

DNARepair

deathgrowtharrest

switchon genes

DNA Repair

-Direct Repair

-Base Excision Repair

-Nucleotide Excision Repair

-Transcription CoupledRepair

-Mismatch Repair

-Recombination Repair

Sunlight

Xeroderma Pigmentosum ~ 1/250,000

DNA Repair Strategies• Direct Reversal

Photolyase, Methyltransferase,Oxidative demethylase

• Excision RepairBase excision, nucleotide excision, mismatch repair

• Lesion AvoidanceTranslesion synthesis, DNA recombination

• Double strand break repairHomologous recombination, Non-homologous end joining

Many genes can influence whether or notan environmental exposure leads to

diseaseEx

posu

re

Expo

sure

--

Envi

ronm

ent

Envi

ronm

ent G

enetic

Genetic

Susceptibility

Susceptibility


Human Health/Disease

Expo

sure

Expo

sure

--

Envi

ronm

ent

Envi

ronm

ent G

enetic

Genetic

Susceptibility

Susceptibility



Sources of DNAAlkylating Agents

Exogenous – outside us

Tobacco Smoke

Fuel Combustion Products

Food Constituents

Food Preservatives

Chemotherapuetic Agents

Endogenous – inside us

S-Adenosylmethionine

Nitrosation of Amines

Lipid Peroxidation

Che

mot

hera

py

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Age

ntA

gent

Genetic

Genetic

Susceptibility

Susceptibility

Treatment RegimenTreatment Regimen

Tumor and normal tissues

CANCER TREATABILITY

Che

mot

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py

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Age

ntA

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Genetic

Genetic

Susceptibility

Susceptibility



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Genetic

Genetic

Susceptibility

Susceptibility



CANCER TREATABILITY

Exp

osur

e

Exp

osur

e --

Env

iron

men

t

Env

iron

men

t Genetic

Genetic

Susceptibility

Susceptibility



Gene-Environment Interaction

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Susceptibility

Susceptibility




Exp

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Susceptibility



Exp

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Susceptibility




Many genes can also influence whether or notcancer chemotherapy is effective

Mouse

Yeast Humans

Bacteria

Yeast Humans

Culturedrodent/human

cells


DNArepair

DNA damage

Cellcyclearrest

How can we identify ALL pathways thatinfluence cell death and mutation?


DNArepair

DNA damage

Cellcyclearrest

Made a start with the buddingyeast Saccharomyces cerevisiae

• Transcriptionalprofiling

• GenomicPhenotyping

Many genomes are now sequenced!!!

5,800genes

sequenced in 1997

14,000genes

sequenced in 2000

~22,500genes

sequenced in 2005

~22,500genes

sequenced in 2005

~22,500genes

sequenced in 2003

4,200genes

sequenced in 1997

E. coli

S. cerevisiae

D. melanogaster

mouse

chimpanzee

human

Made a start with the buddingyeast Saccharomyces cerevisiae

• Transcriptionalprofiling

• GenomicPhenotyping

5,800 genes to analyze!!

No treatment 0.01% MMS

0.02% MMS 0.025% MMS

1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

H

G

F

E

D

C

B

AH

G

F

E

D

C

B

A


0.02% MMS 0.025% MMS

1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

H

G

F

E

D

C

B

AH

G

F

E

D

C

B

A

Transcriptional profiling

Jelinksy and Samson 1999Jelinsky et al 2000

Begley et al 2002Begley et al 2004

Genomic phenotyping

>30% of genome is transcriptionallyresponsive to DNA Damaging agents

>30% of proteins are involved in recoveryafter exposure to DNA Damaging agents

The global or systems approach – studyingall genes and all pathways together


0.02% MMS 0.025% MMS

1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

H

G

F

E

D

C

B

AH

G

F

E

D

C

B

A


0.02% MMS 0.025% MMS

1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

H

G

F

E

D

C

B

AH

G

F

E

D

C

B

A

Transcriptional profiling

Jelinksy and Samson 1999Jelinsky et al 2000

Begley et al 2002Begley et al 2004

Genomic phenotyping

>30% of genome is transcriptionallyresponsive to DNA Damaging agents

>30% of proteins are involved in recoveryafter exposure to DNA Damaging agents

The global or systems approach – studyingall genes and all pathways together

PATHWAYS REPRESENTED

DNA RepairCell Cycle

DNA ReplicationTranscription

SignalingTransport

Protein DegradationProtein Synthesis

Amino Acid MetabolismRNA Metabolism

Cell Membrane BiosynthesisChromatin structure

Of 4,733 strains tested

Sensitive Resistant

MMS: 1441 0

t-BuOOH: 447 78

4NQO: 819 2

UV: 288 1

Assembling the damagerecovery system

Phenotype Data Molecular Interaction Data

Sensitive

No Phenotype

Essential

Protein-Protein

Protein-DNA

The Yeast Protein Interaction Networkviewed in Trey Ideker’s Cytoscape

4,684 proteins connected by 14,993 p-pinteractions

- Essential, Toxicity Modulating (MMS, 4NQO, UV,

t-BuOOH), No Phenotype

Phenotypic Annotation of the InteractomeTom Begley & Trey Ideker

aa

a a

C = 1

C = 0.33

C = 0

C = NA

aaa

a a

C = 1

C = 0.33

C = 0

C = NA

a

Filter out subnetworks with highclustering coefficients –

indicative of protein complexes

A) B)

C) D)Chromatin RemodelingDNA Damage ResponseTranscriptionVacuole FunctionSignal TransductionOtherUnknown Function

MMS 4NQO

UV t-BuOOH

MMS 4NQO UV t-BuOOHMMS 4NQO UV t-BuOOH

E)

RNA Pol IIand mediator

complexSwi/snfcomplex

NERcomplex

Vacuolarsorting

Transc.Reg.

complex

RNA Pol IIC-terminal

kinase

Nuclearpore

complex

Vacuolarmembranecomplex

Maya Said and Tom Begley

DNA Repair deficiency results in strikingdifferential network activation

Repair proficient Treated Repair deficient - Treated

166 nodes/854 edges 11 GO categories

590 nodes/3008 edges21 GO categories

How can we identify ALL pathways thatinfluence cell death and mutation?

DNA repair and cell cycle arrest plusMANY other unexpected pathways also

play a role


DNArepair

DNA damage

Cellcyclearrest

Many genomes are now sequenced!!!

5,800genes

sequenced in 1997

14,000genes

sequenced in 2000

~22,500genes

sequenced in 2005

~22,500genes

sequenced in 2005

~22,500genes

sequenced in 2003

4,200genes

sequenced in 1997

E. coli

S. cerevisiae

D. melanogaster

mouse

chimpanzee

human

David Sabatini, CCR Member,et al., Cell 2006

The expression ofeach mouse or

human gene can bedampened !!! Thisrequires robotics

and highthroughputtechnology

RNAi-mediatedknock down of

gene expression

LITTLE or NO PROTEIN

Che

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py

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Age

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Genetic

Genetic

Susceptibility

Susceptibility



CANCER TREATABILITY

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Age

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Genetic

Genetic

Susceptibility

Susceptibility



Che

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Genetic

Genetic

Susceptibility

Susceptibility



CANCER TREATABILITY

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Genetic

Susceptibility

Susceptibility




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Susceptibility




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Susceptibility

Susceptibility



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Susceptibility




Determine what genes influence cancersusceptibility and whether cancer

chemotherapy will be effective

• The Genome is an extremely large and critical targetfor toxic agents – exposure to which is unavoidable

• Damage to the Genome can result in cell death, smallmutations and large chromosome rearrangements all ofwhich contribute to aging and disease, including cancer

• Sophisticated mechanisms exist to ensure thatdamage in the genome is repaired

• Cells activate a plethora of pathways upon exposureto DNA damaging agents and not doing so can lead todevastating disease

MAJOR POINTS

dna: mutation, repair and the...

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