LEC9 Instability

8/13/2019 LEC9 Instability

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INTRACELLULAR SYSTEMS

ERRORS

producing high-rate mutations


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in germplasm spontaneous abortions

or babieswith genetic syndromes

or just nothing (Inherited:materialfor evolution);

In somatic cellsspontaneous cell deathorimproperly functioning cells(never going to bedetected)

or just nothing (not-inherited:lostin evolution)

or CANCER

De novo mutations in human genes


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Mutations in different places

produce different effects

Microsatellites

2-5 bp repeats

variation in repeat

number

very diverse

Synonymous variation

frequency is several

times higher than

nonsynonymous

no effect on protein

Nonsynonymous

(aa replacement changes)

affect amino acid sequence

Non-coding variation

largely do not affect function

similar frequency as to synonymous

Only a subset of protein variation

affects function or charge

www.stats.ox.ac.uk/~mcvean/DTC/Human.ppt


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How diverse are humans elsewhere in the

genome?

On average, two genomes differ at about 1 in 1000 bases

This figure is lower in coding regions and higher for regions with short

repeated motifs

Mutations in genes can affect function detrimentally Slippage during replication and unequal crossing over

lead to silent variation in repeat number

Humans are one of the least diverse organisms (excepting cheetahs)

Species Diversity (percent)Humans 0.1

Chimpanzees 1

Drosophila simulans 2

E. coli 5

HIV1 30

www.stats.ox.ac.uk/~mcvean/DTC/Human.ppt


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Human autosomal diversity goes back about 1MY

The mutation rate is about 1.5x10-8per site per generation

Diversity of 0.1% implies an average date of 67,000 generations orabout 1.3MY if the average age of reproduction is 20 years (cfchimps)

Different stories from different genomes

The MRCA = Most Recent Common Ancestor The human mtDNA MRCA is estimated to be about 238,000 YA

The human Y chromosome MRCA is estimated to be about 91,000YA (Tang et al. 2002)

Why do different parts of the genome tell different stories?

Chance

Different effective population sizes

Different behaviours of men and women

Natural selectionwww.stats.ox.ac.uk/~mcvean/DTC/Human.ppt


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COMMON TYPES

OF POINT MUTATIONS


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Sources of point mutations in DNA

1) Spontaneous or assisted chemical changes in the DNA

Oxidation (red arrows) Hydrolysis (blue arrows)

Methylation (green arrows)


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Sources of point mutations in DNA

1) Spontaneous or assisted chemical changes in the DNA

Deamination as an example

Hypoxantine;

pairs with CNo deamination

Uracil;

pairs with TXantine;


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Types of mutations that can occur

Point mutations (single nucleotide changes)

TRANSITIONS

TRANSVERSIONS

PURINEPURINE orPYRIMIDINEPIRIMIDINE

purine pyrimidine or

pyrimidine purine

MOSTCOMMON

Pairing is possible

due to tautomeric shifts or

ionizing that allows mispairing

Pairing is energetically infavourable,but Pur-Pur pairs are possible (G-A)


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Other types of mutations that can

occur

2. Small insertions/ deletions of 1-5 bp

3. Large chromosomal deletions

4. Translocations Do not forget about them !

Point mutations much more tolerable

for cell reparation system,

and unlikely to awake apoptosis pathway.

When people talk about carcinogenic mutations,

most often they talk about point mutations.

By Failure to repair

abasic sites


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RATES of molecular events in

human cells

Random gain of mutations

(low-level; natural cause)

Early stages

of natural cancerin elderly;

spontaneous

mutations

in germ plasm

Forced gain of mutations

(median level; X-ray,

chemical carcinogens)

Early stages of cancer

and germplasm in

exposed people;

Very high rate of mutations

(in cells that lost one or more major mechanisms

of DNA repair)

Certain genetic syndromes;late stages of almost any cancer

Rate of mutations in human

genes


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HOW TO CALCULATE RATE

OF MUTATIONS IN HUMAN

CELLS ?

By HPRT assay


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Human HPRT gene

Human lymphocytes

survive treatment

with 6-thioguanine

(poison)

ONLY if HPRT gene is

mutated

-- Located on chromosome X-- Encodes the enzymehypoxanthine phospho ribosyl transferase

-- Normal function of HPRT is metabolic salvage of the purines

(hypoxanthine and guanine) into nucleotides,

inosinic acid, and guanylic acid

6-thioguanine


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HPRT ASSAY

The Hypoxanthine Phosphoribsyltransferase Assay.

6-thioguanine = Poison

6-thioguanine = OK

HPRT +/+

HPRT -/-

So, if cell acquire mutation in

HPRT, it become resistant

to 6-thioguanine compound

We can directly count mutant

coloniesand compare thisnumber with number of cell

seeded on plate


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HPRT mutant frequencies

In peripheral T cells

In 49 healthy,

non-smoking adultsrates

varied from 0.25 to 9.64 x 10(-6).

CYP1A1, GSTM1 and NAT2 polymorphisms

have no influence on HPRT mutation frequencies;

mismatch repair genes was also not damaged

What made mutation frequencies

so different (>10 times)???


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human cells



Early stages


spontaneous

mutations

in germ cells





and germ cells in

exposed people;


(cell lost one or more major mechanisms of DNA repair)Certain genetic syndromes;

late stages of almost any cancer


genes


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Environmental influences on

mutation frequencies?

Yes and noMany reports withcontradictions

Maternal alcohol consumption during pregnancy

leads to increase of Mut freq. in HPRT assayon T-lymphocytes from newborns

Mutat Res 1999 Dec 17;431(2):279-89

Early pregnancy alcohol RR = 1.84

During pregnancy alcohol RR = 2.99

Smoking during pregnancyhave an influence

on mutational spectrum,

but not on the their frequency


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Mutations in HPRT and smoking

Yes, smoking increases mutation frequency

No, smoke does not have any influence

A comparison of mutation frequenciesin the K-ras, p53 and HPRT genes

between the normal lung tissueof smokers and non-smokers

indicates that the rate of mutation in smokers

is only ~1.6 fold higher than in non-smokers


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HPRT rates among

Radiationexposed peoples

Among Hiroshima-Nagasaki survivors (43 Rad in average)

1 mut per 10-8 per base pair per generation

indistinguishable from that of Japanese controls

Chernobyl clean-up workers:

40% increase in mutation rate

in the first year after accident, .then it declined.

So, for lymphocytes classical HPRT

assays

do not reveal strong mutational load


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Mutagens = (Carcinogens)

Any agent that produces mutations, e.g.

tobacco smoke,

certain industrial chemicals,

ionizing radiation

(such as X-rays and ultraviolet rays).


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Strong envinronmental carcinogens

ethylene oxide ; 1,3-butadiene ; benzene

JUST marginal increasein HPRT mutability

when measured asin vivoexposure(on plant workers populations)

In the same timecell line-based or

mice/rat-based assays with

direct addition of carcinogenshow

strong increasein HPRT mutability


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polycyclic aromatic hydrocarbons (PAHs) derivates

can bind and damage DNA

In the Liver

Oxidized form of bp


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certain strains of the fungi

Asp ergi l lus f lavus

and A. parasi t icus

oltipraz,an inducer of Phase 2metabolizing enzymes,

significantly increased biomarkers

of aflatoxin detoxification

Prevention trial in China

Aflatoxin B1


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human cells



Early stages


spontaneous

mutations

in germplasm





and germplasm in

exposed people;


(cell lost one or more major mechanisms of DNA repair)Certain genetic syndromes;

late stages of almost any cancer


genes


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Nature Reviews Cancer3; 155-168 (2003); doi:10.1038/nrc1011ATM AND RELATED PROTEIN KINASES: SAFEGUARDING GENOME INTEGRITY

Repair Give up

Amount and type

of damage

can be handled

Damage is excessive

and/or irreparable

Activation of the survival

response network

Activation

of the apoptosis

Low fidelityrepair

CANCER CELL DEATHCELL

SURVIVAL


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DNA repair

1/1000 bp of newly synthesized DNA is incorrect

but most of mutations are fixed on spot

Only 1/1,000,000 bp are actually miscopied(because of help of DNA repair mechanisms)

Loss of DNA repair mechanisms results ingenomic instability, resulting in massive

amount of genetic mutations

Many syndromes connected to mutations


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Many syndromes connected to mutations

in reparation-related genes

are associated with increase in cancer incidence

Physiological importance of


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Physiological importance of

checkpoint pathwaysATM (ataxia telangiectasia) as example

loss of motor control owing to Purkinje cell loss,

masked faces; oculomotor apraxia

Immune deficiencies:

High frequencies of cancer:

www.neuro.wustl.edu/neuromuscular/ ataxia/dnarep.html

1 in 40,000-300,000 live births:Ataxia:

Telangectasias:

Dilated small blood vessels; Skin & Ocular; Onset 4 to 6 years

Recurrent respiratory infections;T-cell function is reduced

14q+ leukemia (T-CLL)

B-cell lymphoma

Why ATM defect leads not only to cancer
http://www.neuro.wustl.edu/neuromuscular/ataxia/dnarep.htmlhttp://www.neuro.wustl.edu/neuromuscular/ataxia/dnarep.html


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Why ATM defect leads not only to cancer,

but also toorganismal defects?cells lacking ATM fail to execute

many of the cellular responses to DNA damage.

DSBs in normal VDJ recombination

and in meiosis also needs repair.

BUT even without any DNA-damaging agent:

3.

during normal cell duplication

numerous chromosome defects occur

(DNA replication errors, such as double-strand breaks (DSBs))

stalledreplication forks

1. 2.

(Cells function in highly reactive

chemical environment)

ATM in oxidative stress

long-lived, terminally differentiated cells

such as Purkinje cells are damaged


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Concepts of checkpoint

DNA damage checkpoints

is aNON-ESSENTIALregulatory pathways

that control the ability of cells to arrest the cell cycle

in response to DNA damage,allowing time for repair.

1. Historical

Several checkpoint genes (ATR, CHK1) are essential for cell and organism survival

Checkpoint pathways are not only

surveyors of occasional damage,

but are

firmly integrated components of cellular physiology.

2. Modern


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Damage checkpoint pathways

control telomere length

induce of cell death by apoptosis

activate DNA repair pathways

Activate different

transcriptional programms

Control the movement of

DNA repair proteins

to sites of DNA damage

Control cell-cycle arrest


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Kevin Springet al., Nature genetics 2002

Survival of ATM +/- and ATM-/- cellsafter IRHUMAN MICE

ATM patient

+/- parents of ATM patient

Normal lymphocytes

ATM -/- mice

ATM +/- mice

Normal mice

ATM i t T S G


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ATM is a true Tumor Suppressor Gene

ATM itself is 1 in 40,000-300,000 live births

(different for different nations)

Heterozygotes in populations: 0.35% to 1%

Breast cancer susceptible

(9-16-time increase in comparison to population)

60% of women withthe ATM gene mutation

will develop cancer

by the age of70.

ATM carriers develop breast cancer in response to radiation

(due to enchanced radiosensitivity of the cells)

Tuberculosis screening and mammogramms

are harmful for ATM carriers

ATM t ti i


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locus.umdnj.edu/nigms/ charmut/atmut.html

ATM mutations in

Ataxia-telangioectasia syndrome
http://locus.umdnj.edu/nigms/charmut/atmut.htmlhttp://locus.umdnj.edu/nigms/charmut/atmut.html


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BIN-BING S. ZHOU AND STEPHEN J. ELLEDGE

BRCA1

tumor suppressor p53

NBS1

Chk2, which is involved

in control of both the G1/S andG2/M cell-cycle checkpoints

SUBSTRATES

ATM acts like any normal kinase

(via phosphorylation)

This processes is similar in their basics

non homologous end joining machinery (NHEJ)


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Homologous recombination

ResectionRad50, Mre11,

Xrs2 complex

Strand invasion

Rad52

Rad51; BRCA2

DNA synthesis

Ligation, branch migration,

Holliday junction resolution

DSB

Non-homologous end-joining

(NHEJ)

Ku70, Ku80

Ligation

DSB

DNA-PKcs

XRCC4/Ligase IV

complex

V-SEGMENT D-SEGMENT

RECOMBINATION

APPARATUS

(RAG1, RAG2)

GENERATION OF DSBs

CODING JOIN SIGNAL JOIN

PROCESSING, LIGATION BY

NHEJ SYSTEM

V(D)J recombination

This processes is similar in their basics

RECRUITMENTof NBS1

RECRUITMENT

Of XRCC4 and

DNA ligase

http://www.welc.cam.ac.uk/~spjlab/M7_SPJ_NHEJ/27

non-homologous end-joining machinery (NHEJ)

Rad50, Mre11,NBS1 complex


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Multiple potential roles for

Ku/DNA-PKcs in NHEJ



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MRE11 geneChromosome 11q21

with milder course of disease

Cerebellar degenerationpresent

Mutated in Ataxia-telangiectasia-like disorder (ATLD)

Unfortunately, DNA-PKs

are not activators of the

global DNA damage response(they respond only on DS breaks)

It is biochemically difficult to draw the line

between sensors and effectors


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Multiple potential roles for

Ku/DNA-PKcs in NHEJ


Nijmegen breakage syndrome


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Nijmegen breakage syndrome(NBS1 -/- defects)

spontaneous chromosomal instability,

sensitivity to ionizing radiation (IR),

and radioresistant DNA synthesisCancer predisposition

(leukaemia, lymphoma, neuroblastoma)

Immune deficiencies

Growth retardation, intellectual impairment

130 cases found worldwide

50-fold risk of early-onset common cancers

Cellulardefects:

NO clinical hallmarks of AT:

(no cerebellar ataxia, no telangiectasia,

no elevated serum alpha-fetoprotein levels

Gonadal failures

LEC9 Instability

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