DNA-Damage

Single strand breaks

Small adducts
Intra-strand crosslinks
Bulky adducts
Double strand breaks
Pyrimidine dimers
DNA-protein crosslinks
Intercalation
Mutasi
C U
 Mutasi, mutan & mutagen
Mutasi
• Perubahan basa dalam sekuen DNA (Pd umumnya
di dalam suatu gene).
• Perubahan ini termasuk : substitusi basa,
adisi, penyusunan ulang dan delesi
Mutan
• Organisme yang mengalami mutasi.
• Mutasi tersebut tentunya terjadi dalam gena yang
menyebabkan terjadinya perbedaan yang nyata
dengan bentuk normal (Wild-Type).
Mutagen
• agen fisika atau senyawa kimia yang menyebabkan
mutasi.
Mutagenesis
A spontaneous mutation occurs once in 108 cells.

The mutation rate can be increased by exposing cells

to mutagens, which are either chemicals or physical
agents such as UV-irradiation.

Both chemicals (EMS) and physical agents act by

causing genetic damage that results in base changes
in DNA
 Causes of DNA Damage: Endogenous & Exogenous
Radiation
Ionizing radiation (, x-ray;  particle)
double-strand breaks with 3’phosphates
UV radiation
pyrimidine dimers (thymine dimers, thymine-cytosine dimers)
Oxidants
Reactive oxygen species (H2O2 , O2- ), often generated by respiration
8-oxo-dG, 2-OH-dA
Chemical mutagens:

Alkylating agents (e.g., ethyl- and methyl-methane sulfonate: 3-Me and 3-ethyl-dA)
Nucleophiles (e.g., hydroxylamine, catalyzes cytosine deamination)
Cross-linkers (e.g. psoralen)
Intercalators (e.g., acridine orange, ethidium bromide):
cause deletions & insertions (i.e., frame-shifts) during replication
Spontaneous deamination: uracil, xanthine, hypoxanthine: repaired by excision repair or
by glycosylase (no xanthine-specific glycosylase)

Replication errors: misincorporation resulting in mismatched base pairs

 Mismatches  saat DNA replikasi

Mutan
Replikasi 5’-ATGGG-3’
3’-TACCC-5’
5’-ATTGG-3’ 5’-ATGGG-3’
3’-TAACC-5’ 3’-TAACC-5’
Normal
5’-ATTGG-3’
3’-TAACC-5’
Perubahan struktur  Mutasi DNA
Mutan
5’-ATXGG-3’
Kerusakan
5’-ATXGG-3’ 3’-TAGCC-5’
nukleotida 3’-TAGCC-5’
5’-ATXGG-3’ 5’-ATCGG-3’
3’-TAACC-5’ 3’-TAGCC-5’
5’-ATTGG-3’ Mutan
3’-TAACC-5’
Normal
 Tipe mutasi
Mutasi pada tingkat DNA
1. Point mutation
a. Substitusi satu basa dengan nukleotida yang lain
2 tipe:
Transisi – perubahan purine ke purine (A to G, G to A)
atau pirimidin ke pirimidin (C to T, T to C)

Transversi – perubahan purine ke pirimidin atau

sebaliknya, e.g. A to C or T, C to A or G

b. Insersi atau delesi

Penambahan atau pengurangan satu base-pairs.
Tipe mutasi
Inversi
Pemotongan sebagian DNA dan kemudian disisipkan
pada tempat yg sama tetapi pada orientasi yg
berbeda
5’-ATTGG-3’ 5’-ATCAG-3’

3’-TAACC-5’ 3’-TAGTC-5’
 Reversion
This is the reverse process of mutation and involves a
mutant regaining a wild-type phenotype, either
through:
1. A back mutation (direct reverse of the
mutation)
2. A reverse or suppressor mutation (not a direct
reverse, but a mutation at a second site
generally within the same gene which
suppresses the effect of the first
mutation). The mutant is described as a revertant.

A practical example of reversion is the Ames test

which is used to test for carcinogens.
What is reversion?
WT, active
Mutate - to + Mutant, inactive
+- +
+

Mutate + to -

+-
-+

WT, active WT ?, active

Same site revertant different site revertant
 Base-pair and Frameshift Mutations

Mutation type

THE NUN SAW OUR CAT EAT THE RAT

THE NSN SAW OUR CAT EAT THE RAT Nonsense

THE SUN SAW OUR CAT EAT THE FAT Missense

THE NUS AWO URC ATE ATT HER AT Frameshift

 Mutasi pada aras gena
1. Silent mutation

Perubahan basa tidak meyebabkan perubahan pada kodon

5’ ATG GGA GCT CTA TTA ACC TAA 3’

met gly ala leu leu thr stop

Silent mutation
( & transition)
5’ ATG GGA GCT CTA TTG ACC TAA 3’
met gly ala leu leu thr stop
 Mutasi pada aras gena

2. Missense mutation
Perubahan basa  Perubahan kodon

5’ ATG GGA GCT CTA TTA ACC TAA 3’

met gly ala leu leu thr stop
Missense mutation
( & transversion)
5’ ATG GGA GCT CTA TTT ACC TAA 3’
met gly ala leu phe thr stop
 Mutasi pada aras gena
3. Nonsense mutation
Perubahan basa  menjadi stop kodon

5’ ATG GGA GCT CTA TTA ACC TAA 3’

met gly ala leu leu thr stop
Nonsense mutation
( & transversion)

5’ ATG GGA GCT CTA TGA ACC TAA 3’

met gly ala leu stop
 Mutasi pada aras gena
4. Frameshift mutation
Hilangnya satu basa  perubahan pada pembacaan

5’ ATG GGA GCT CTA TTA ACC TAA 3’

met gly ala leu leu thr stop

Frameshift mutation

5’ ATG GGG AGC TCT ATT AAC CTA A 3’

met gly ser ser ile asn leu
Apa akibatnya jika
 Delesi 1, 2 atau 3 basa
 Adisi 1, 2 atau tiga basa
 Substitusi 1, 2 atau 3 basa
 Baik berurutan maupun tidak berurutan
DNA T RNA U
Untuk menaikkan kebenaran
pesan genetik

 URACIL DNA GLYCOSYLASE

 ENZIM MENGHIDROLISA IKATAN
GLIKOSIDIK ANTARA U DAN GULA
Human Base Excision Repair Mechanism

5‘

Glycosylase

5‘

Polymerase

5‘
 Direct Damage Reversal:
correction of pyrimidine dimers

UV irradiation (200-300 nm)

causes formation of pyrimidine
dimers in which consecutive
pyrimidine residues are linked
by a cyclobutane ring:

Pyrimidine dimer formation can

be directly reversed by
Photolyase which absorbs
photons (300-500 nm) and
catalyzes reversal of the photo-
cyclization reaction.

cyclobutane ring in thymine dimer

Nucleotide excision repair in Eukaryotes
Multi protein complex (> 16 proteins)

Deficiency of NER are manifested by diseases:

Xeroderma pegmentosum - inability of skin cells to

repair UV-damadge
Individuals with this disease are extremely sensitive
to UV-light, higher level of skin cancer

Cockayne syndrome – defects in same genes as

Xeroderma pegmentosum + two additional genes.

Sensitivity to UV-ligh, neuron denyelination, normal

level of skin cancer.
Correction of pyrimidine dimers by
DNA photolyase

hu(300-500nm)
Pyrimidine dimer

chromophore
FADH -

Pyrimidine monomers

Base flipping
Fotoliase
Regulasi Repair:
SOS Regulon
Mismatch repair- colon cancer
 Penyebab hereditary nonpolyposis colon
cancer (HNPCC)
 Mutasi pada mikrosatelit –hMLH1, hMSH2 =
MutS dan MutL pada E.coli

 CC 4.8
 Mismatch repair
 Correct insertion or deletion up to 4 nt
 Additional proofreading after Pol
E. Coli
DNA methylation site: A base in GATC
sequence
Newly synthesized DNA – hemimethylated

MutS, MutL and MutH protein complex

participate in the mismatch repair
MutS recognize mismatch
MutS2MutL2 recognize methylated GATC
palindrome, and activates
endonuclease MutH – nick DNA

The unmethylated ssDNA is removed by

UvrD protein

The gap is filled by DNA polymerase

Human DNA:
Methylated in C position in CG sequence
Homologs of MutS and MutL proteins (MSH
and MLH) proteins
Beda untai lama dan baru ?
 HUNTINGTON DISEASE

CAG : GLUTAMIN
NORMAL
PENDERITA 36 -82 KOPI
HUNTINGTON DISEASE
 CAG : GLUTAMIN

 PENDERITA 36 -82 KOPI

Fagile X syndrome: berpengaruh pada sistem

regulasinya
Double Strand Break Repair
Single Strand Annealing
- resection of 5‘-ends allows formation of joint molecules
with homology regions
- DNA synthesis, removal of non-homologous ends, ligation
- introduction of deletion

Non-Homologous End-Joining (NHEJ)

- probably DNA-dependent kinase (DNA-PK) aligns broken ends
- ligation by DNA ligase IV
 Normal Base Pairing and Mispairing
H

CH3
O H
O H H N N O
N
dRib H
N N O N
H N O N
dRib N
O H
O N N N N
N
dRib
dRib H
Deoxythymidine : Deoxyadenosine Deoxycitidine : Deoxyguanosine
CH3
O
O
CH3
N N O
H
dRib
O N
O N
H
N N
N
dRib
H

Deoxythymidine : Alkyl deoxyguanosine

 Possible Consequences of DNA-Adducts
A T
Deoxythymidine : Alkyl deoxyguanosine T A
CH3
aG T
O
O A T
C G
CH3 T A
N N O
dRib
H aG T A T
O N
O N
C G T A
H
N N
N
dRib
A T
H
C G
A T A T
T A T A A T
G C aG C T A
C G C G G C
A T
C G
T A
G C A T
Methylbromide
C G
T A
G C
C G
MUTAGEN
MUTAGEN KIMIAWI
 BASA ANALOG
 5-Bromourasil dan 2-aminopurin bergabung pada
DNA seperti asam nukleat lainnya
 BU = analog timin, yang berpasangan dengan A
tetapi juga dapat berpasangan dengan G
 Terjadi mutasi dari A:T menjadi G:C
BROMURASIL DALAM BENTUK ENOL BU:G
AFLATOKSIN B1
 AFLATOKSIN B1 OLEH SITOKROM P450
DIUBAH MENJADI SENYAWA EPOKSID YG
BEREAKSI DENGAN G

 G:C MENJADI T:A

SENYAWA INI BEREAKSI
DENGAN 7-N GUANOSIN
menghasilkan radikal

G:C MENJADI T:A

Aflatoxin B1 exo-8,9-Epoxide forms DNA-Adducts
O
O
O HO O
O H
O
O H O
HN N O
P450 O
+
N H O
OCH 3
NH 2 N
H O
O OCH 3 dR
O
DNA DNA
H O

O exo-8,9-epoxide DNA-Adduct (N7-dGuo)

H O OCH 3

Aflatoxin B1 O
O
O
O
SG
P450 O O HO H
O
H GST
O
O
H O
O OCH 3
H OCH 3

endo-8,9-epoxide
ASAM NITRIT
 MENGOKSIDASI ADENIN (A) MENJADI
HIPOSANTIN
 HIPOSANTIN INI AKAN BERPASANGAN
DENGAN C

 A:T MENJADI G:C

A:T G:C
SENYAWA INI INTERKALASI PADA DNA
MENYEBABKAN INSERSI ATAU DELESI PADA
WAKTU REPLIKASI
Ames Assay

- Mutagenicity test

- Point mutations

- Backward mutations

- Direct mutagens

- Indirect mutagens
The “Ames Assay” Tester Strains
strain point mutation LPS repair R-factor
---------------------------------------------------------------------------------------------------------------------
TA97 hisD6610 frameshift (+1, G:C) rfa uvrB pKM101
hisD1242 frameshift (+1, G:C)

TA98 hisD3052 frameshift (-1, G:C) rfa uvrB pKM101

TA100 hisG46 substitution (G:C) rfa uvrB pKM101
TA102 hisG428 substitution (A:T) rfa + pKM101
(paQ1)

rfa partial loss of the lipopolysaccharide barrier

uvrB deletion of a gene coding for a DNA repair system
pKM101 plasmid, increases mutagenensis by enhancing an error prone DNA repair system
(carries ampicilline resistence gene)
paQ1 plasmid, carries the hisG428 mutation ( carries tetracycline resistence gene)
Standard Test Set
- 4 Strains of S. typhimurium: TA97, TA98, TA100, TA102
- Metabolic activation (-/+ S9 mix)
- At least 3 concentrations of test substances
- At least 2 plates per concentration
(duplicates or better triplicates)

- Positive / negative controls

- Independent repeats of the entire experiment
(3x => 6values per concentration of substance)

A substance is considered to be mutagenic if

- number of revertants are at least doubled
by the test substance
- a (linear) dose-response-relationship observed
“Successfull Ames-Test”

- NO contamination with other bacteria or fungi etc.

- Healthy background lawn
- Spontanous revertant rate is in typical range of individual
strains (variations between labs occur):
TA97 => 90 - 180
TA98 => 30 - 50
TA100 => 120 - 200
TA102 => 240 - 320

- Pos and neg controls should be pos and neg respectively

 Gene A Chromosome 1

Gene B Chromosome 2
Fusion Gene

Primary transcript

Fusion mRNA

Unique Properties Altered Pattern of gene expression

Chimaeric Acts as an oncogene

protein
Differentiation Blocked

65% of leukaemias are characterised

by particular somatically acquired Continued self-renewal
chromosome translocations
 Bcr-abl = constitutively
active tyrosine kinase

(The protein product from this fusion

gene only found in ~70% of patients)

Chronic myeloid leukaemia (CML) is characterised by

the t(9;22)(q34;q11) reciprocal translocation
Deteksi fusi gen- leukemia
 Translokasi kromosom 9 dan 22 pada q34
dan q11
 Translokasi kromosom 11 dam 17 pada q 22
dan q21
 Bagimana cara mendeteksinya ?
 Chemical Carcinogenesis

Initiation Promotion Progression

Mutation

Exposure DNA-Damage Initiated Preneoplasic Cancer

cell clone

Excretion DNA-Repair
Apoptosis
Exposure Effect
<1 day 20-40 Years
Initiation and Promotion in Carcinogenesis

Application of
no tumour initiating
or
tumour promoting
substance
no tumour

Time period
no tumour
Time

no tumour
Emergence of a cancer cell
 Metabolism of NNK,
a Tobacco-specific Nitrosamine

O N O
N
CH3
NNK-N-oxide NNAL NNAL-Gluc
N NNK
Cytochrome
P450
NNAL-N-oxide Urine
[-Hydroxylnitrosamine]

Aldehyde + Diazohydroxide

DNA-Adducts Mutations: G->A

G->T
Test Systems:
Mutagenicity tests:
- COMET- Assay (indicator test)
- HPRT -Test (gene mutation)
- Micronucleus Test (chromosomal mutation)

Promotion tests (mechanism based):

- HPRT-Test (cell-cell-communication)
- Use of particular animals (2m-globulin)
- Induction of peroxisome proliferators
.....
COMET Assay:
Single cell gel electrophoresis assay

Determines DNA damage and/or repair in

individual eukaryotic cells
=> Indicator assay

- In vitro & in vivo

- Measures the size of DNA fragments

DNA damage has to be converted into DNA fragments
(by pH or lesion specific glycosylasese/endonuclease treatment)

- DNA fragments or relaxed chromatin migrate away from

nucleus during gel electrophoresis
COMET-Assay: Principle of single cell gel assay
Single strand break (SSB) damaged
DNA
alkaline labile site
alkaline pH

SSB
unwound DNA
SSB

agarose gel electrophoresis

+ -

normal nucleus

damaged nucleus
COMET-Assay: Principle of single cell gel assay

- Exposed cells are embedded in agarose on

microscopic slides
- Cell lysis to remove all proteins
- DNA unwinding under alkaline/neutralpH- Electrophoresis
- DNA is stained with flourescent dye
- Evaluation under microscope
- Scoring: number of damaged cell and
severity of damage (Type I to V) in x cells/1000 cells
- Refined scoring by determination of e.g. tail length
COMET-Assay: Representative Photomicrograph

Damaged cells Undamaged cells

Strand breaks increase DNA migration!

Cross-links produce decrease in DNA migration!

Representative photographs of comets from murine fibroblasts treated with 50mM H2O2.
Lysis: pH 10; unwinding: pH 13, 20 min; electrophoresis: pH 13, 15 min.
Source: COMET Assay Interest Group h( ttp:// www. geocities. com/ cometassay/ introduction. htm)
 Lead Chloride Induces Micronuclei

Positive controls: Test substance:

Methyl methane Vincristine: Lead chloride:

sulfonate: aneugenic effect aneugenic effect
clastogenic effect

This has to be verified by kinetochore staining!

 Chemoprevention

M.B. Sporn, 1976:

Chemoprevention means the use of specific,
natural or synthetic chemicals in order to
reverse, suppress or prevent the development
of carcinogenic progression into malignancy.