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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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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 cells
Forced gain of mutations
(median level; X-ray,
chemical carcinogens)
Early stages of cancer
and germ cells in
exposed people;
Very high rate of mutations
(cell 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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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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RATES of molecular events in
human cells
Random gain of mutations
(low-level; natural cause)
Early stages
of natural cancerin elderly;
spontaneous
mutations
in germplasm
Forced gain of mutations
(median level; X-ray,
chemical carcinogens)
Early stages of cancer
and germplasm in
exposed people;
Very high rate of mutations
(cell 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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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.html8/13/2019 LEC9 Instability
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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.html8/13/2019 LEC9 Instability
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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
http://www.welc.cam.ac.uk/~spjlab/M7_SPJ_NHEJ/27
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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
http://www.welc.cam.ac.uk/~spjlab/M7_SPJ_NHEJ/27
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