PERUBAHAN SUSUNAN NUKLEOTIDA PADA DNA

PURNOMO SURYOHUDOYO
EMA QURNIANINGSIH

DEPT. OF BIOCHEMISTRY
SCHOOL OF MEDICINE
AIRLANGGA UNIVERSITY
 1901 De Vries
 Any heritable changes of
phenotypic characters Mutation
 New species were believed to
arise by single mutational events

 1910  Thomas Morgan &

colleageus
 Systematic study of mutation by genetic
analysis
 1927  HJ Muller
 Induced mutation in drosophilla by x-rays
MUTASI
Merupakan perubahan pada susunan nukleotida DNA

Dapat terjadi pada tingkat :

• Nukleotida
• GEN
• Kromosom
(aberasi kromosom)

Mutasi yang terjadi pada sel somatik, tidak diturunkan

pada generasi selanjutnya.
Mutasi yang terjadi pada sel gamet, bisa diturunkan pada
generasi selanjutnya.
1. TINGKAT NUKLEOTIDA
Dapat menyebabkan perubahan struktur protein : tanpa
atau dengan perubahan fungsi

2. TINGKAT GEN
Duplikasi Gen
Delesi Gen
Gen Gabungan
3. KROMOSOM
Melibatkan banyak gen
Delesi
Duplikasi / Multiplikasi
Inversi
Translokasi
 MUTASI TINGKAT NUKLEOTIDA (1)
1. MUTASI TITIK ( POINT MUTATION )
Penggantian 1 nukleotida dengan nukleotida lain
 MUTASI TINGKAT NUKLEOTIDA (2)

2. INSERSI ( INSERTION )

Penambahan satu atau

lebih nukleotida

3. DELESI ( DELETION )

Penghapusan satu atau

lebih nukleotida
 MUTASI TITIK ( POINT MUTATION )
• Penggantian 1 nukleotida dengan nukleotida lain

1. TRANSISI
Purin  Purin
Pirimidin  Pirimidin
A G

T C

2. TRANSVERSI
Purin Pirimidin
T A

C G
CONTOH – CONTOH MUTASI TITIK

UUG leu Tak terjadi perubahan Asam Amino

 Mutasi tersembunyi (silent mutation)
UUA leu
“Silent” mutation : does not change an amino acid, but in some cases can still
have a phenotypic effect, e.g. by speeding up or slowing down protein
synthesis, or by affecting splicing.

UUG leu UUG leu UUG leu

UUC phe UCG ser GUG val

Terjadi perubahan asam amino yang dapat (tidak selalu)
mempengaruhi fungsi protein

- Fungsi tak terganggu Acceptable missense

- Fungsi terganggu  Partially acceptable missense
- Tak berfungsi  Unacceptable missense
• Conservative missense mutation : result in amino acid
change, but the properties of amino acid remains the same.

• Non-conservative missense mutation : result in amino acid

change that has different properties than wild type.
 CONTOH – CONTOH MUTASI TITIK

AUG Sandi Inisiasi

Tak terjadi translasi protein kecuali bila di daerah hilir
AUA kebetulan ditemukan urutan AUG lain  umumnya
menghasilkan protein yang tak berfungsi

CAA gln

AUU Sandi Terminasi

Terjadi terminasi dini (premature termination)  protein
yang tak berfungsi / terganggu fungsinya

UAG Sandi Terminasi

Terjadi pembacaan berlanjut (readthrough)  protein
UCG trp yang tak berfungsi / terganggu fungsinya
CONTOH AKIBAT MUTASI TITIK
HEMOGLOBINOPATI
1. Fungsi tak terganggu

2. Fungsi terganggu

3. Tak berfungsi
 Dikaitkan dengan fungsi protein,
mutasi bukan hanya menyebabkan
“loss of function” tetapi juga bisa “
gain of function”.
 Loss of function terjadi pada gen dengan
fenotip resesive, kecuali organisme
haploid dan haploinsufisiensi.
 Gain of function terjadi pada gen dengan
fenotip dominan.
 DELESI & INSERSI

DELESI
 DELESI & INSERSI
INSERSI
INTRON MUTATION

DONOR
SPLICE SITE
ACCEPTOR
SPLICE SITE
DNA
NORMAL SPLICING

mRNA

DONOR SITE ABOLISHED

EG. : GT  AT
ABNORMAL SPLICING

EXTRA SPLICE SITE GE

GENERATED AT 
EG : GC  GT
Normal ALTERNATIVE SPLICING

Abnormal

Abnormal mRNA is usually degraded by nucleases

 MUTASI PADA TINGKAT GEN

 Timbul karena terjadi persilangan tak seimbang

(Unequal Cross – Over) antara 2 kromatid saat
meiosis

 Terjadi Delesi dan Duplikasi Gen atau timbul Gen

tergabung (Fused Gene)

Contoh : THALASEMIA 
UNEQUAL CROSS - OVER

Equal cross-over

Unequal cross-over
Duplication of A1
Deletion of A1

Unequal cross-over
Fused genes
(BA)
Partial deletion of A
Complete deletion of B
HEMOGLOBINO PATI
THALASSEMIA

 THALASSEMIA

 THALASSEMIA - 1

 THALASSEMIA – 2
TRANS (BLACKS)
 THALASSEMIA – 2
CIS (ASIANS)
Hb H DISEASE

Hydrops Foetalis ( † )

 THALASSEMIA
UNEQUAL CROSS OVER
OF TRINUCLEOTIDE REPEATS

UNEQUAL CROSS OVER

CONTRACTION

EXPANSION

TRINUCLEOTIDE REPEAT
Trinucleotide repeat when expanded can cause disease,
mostly with neuromuscular symptoms
TRINUCLEOTIDE REPEATS

CAG (Gln) Huntington disease

spinal & bulbar muscular
1 atrophy
spinocerebellar ataxia
CTG (Leu) Myotonic dystrophy
GAA (Glu) Freidrichs ataxia
CGG (Arg) Fragile x syndrome A
(Frax A)
GCC (Ala) Fragile x syndrome E
(Frax E)
ABERASI KROMOSOM
NORMAL

DELESI TERMINAL

INTERCALARY

DUPLIKASI
HOMOGENEOUSLY
STAINING REGION

DM : DOUBLE MINUTE
TRANSLOKASI

INVERSI
 CONTOH : NEOPLASMA YANG MENUNJUKKAN
ABERASI KROMOSOM
NEUROBLASTOMA DEL (1) (p31 : p36)
SMALL LUNG CA DEL (3) (p14 : p23)
WILM’S TUMOUR DEL (11) (p13)
RETINOBLASTOMA DEL (13) (q14)
ACCUTE MYELOID LEUKEMIA DEL. LONG ARM 7
CHRONIC MYELOID LEUKEMIA t (9, 22) (q34, q11)
BURKITT’S LYMPHOMA t (8,14) (q24, q32)
t (2,8) (p12, q24)
t (8, 22) (q24, q11)
ACCUTE PROMYELOCYTIC
LEUKEMIA
HL 60 HSR, DM

FRANKS & TEICH

“ Introduction to the cellular & molecular biology of cancer”

Oxford .U. Press, 1986

MUTAGENESIS
MUTASI
Dapat terjadi :
1. Secara Spontan
A. Kesalahan replikasi :
Salah pasang Basa (mismatch), adisi dan delesi
B. De Purinisasi (A, G)
Ikatan basa purin relatif lemah
 mudah lepas
 “Apurinic Sites”
C. Deaminasi (A,G,C)
Gugusan NH2 mudah lepas melalui hidrolisis
C  U
G  Xantin (X)
A  Hipoxantin (HX)
2. Akibat bahan kimia
“ Chemical Mutagenesis”
3. Akibat Penyinaran
“ Physical Mutagenesis”
Sinar U.V.
Sinar X & 
 MUTASI KARENA “MISMATCH
1. ISOMER ABNORMAL
TAUTOMER :
Tautomers are two molecules with the same molecular formula but
different connectivity - constitutional isomers (positions of atoms and
bonds between them), in other words - which can interconvert by
simple movement of electrons and atoms.

- KETO ENOL
- AMINO IMINO

ROTAMER : Terbentuk karena putaran tunggal antara

molekul gula (deoksi ribosa) dan basa purin (adenin dan
guanin)

- ANTI SYN
2. PASANGAN GOYANG (“WOBBLE”)
Common forms Rare forms

Rare forms

Common forms

https :// nature.com

The disposition of
binds is very
important to the
hydrogen bond
presentation;
• N3 is a
hydrogen bond
Acceptor in
cytosine
• and a Donor in
uracil/thymine.
TAUTOMER
SITOSIN (C)
Tautomer
Imino C
Mirip T
Dapat berpasangan
dengan A

TIMIN (T)
Tautomer
Enol T
Mirip C
Dapat berpasangan
dengan G
MUTATION DUE TO ABNORMAL TAUTOMER

BEFORE DNA

mRNA
PROTEIN

AT  GC
Mutation

AFTER DNA

mRNA
PROTEIN
HIS  ARG
1. 1st Replication
2. Accidental incorporation of abnormal (imino) tautomer of C (C*)
on – strand
3. 2nd replication
4. Abnormal Tautomer on – strand assumes its normal (amino)
tautomer (C*  C)
5. Incorporation of G on + strand
 ANTI
Pu
Ri

SYN
Pu
Ri

ROTAMER
Nitrogen atom
ROTAMER

A ANTI
Basa Adenin (A) dalam
bentuk normal (anti)
seharusnya berpasangan
dengan basa Timin (A-T),
A SYN membentuk A-A dengan A
(syn)

G ANTI
Basa Guanin (G) dalam
bentuk normal (anti)
seharusnya berpasangan
dengan basa Citosin (G-C),
membentuk G-G dengan G
G SYN (syn)
MUTATION DUE TO ABNORMAL ROTAMER
BEFORE DNA

mRNA
PROTEIN

GC  CG
Mutation

AFTER DNA
mRNA
PROTEIN
ASP  HIS
1. 1st Replication
2. Accidental incorporation of abnormal (SYN) Rotamer of G (G*)
3. 2nd replication
4. Abnormal Rotamer on – strand assumes its normal (anti)
Rotamer (G*  G)
5. Incorporation of C on + strand
PASANGAN GOYANG
( “WOBBLE” )

G G - G

G - A
G
ATOM N

A SCIENCE VOL. 273 p: 1548

13 – 9 - 1996
 Figure 2: Wobble in mismatched nucleotide base pairs.
A shift in the position of nucleotides causes a wobble between a normal thymine and
normal guanine. An additional proton on adenine causes a wobble in an adenine-
cytosine base-pair. It occurs because the DNA double helix is flexible and able to
accommodate slightly misshaped pairings.

© Nature Education Adapted from Pierce, Benjamin. Genetics: A Conceptual Approach, 2nd ed. All rights reserved
DEAMINASI
TAUTOMER U (KETO)
MIRIP T
DAPAT BERPASANG
SITOSIN (ENOL) URASIL (KETO)
DENGAN A
(C) (U)

(ENOL) (KETO)
ADENIN HIPOXANTIN
(A) (HX)
TAUTOMER HX (KETO), MIRIP G
DAPAT BERPASANGAN DENGAN C

(ENOL)
GUANIN XANTIN (KETO)
(G) (X)
TAUTOMER X (KETO), MIRIP A ATOM N
DAPAT BERPASANGAN DENGAN T
DEPURINISASI

DEPURINISASI

REPLIKASI UTAS A

TERJADI DELESI Terjadi pemasangan secara acak

 MUTASI TITIK
(Bila yang dipasang A, G atau T)
MUTAGENESIS
KARENA BAHAN KIMIA

1. “NON ALKYLATING AGENT”

• Deaminasi (HNO3, H2SO3)
C  U
G  X A  HX
• Mengubah tautomer yang dominan
• “ambiguous behavior”
2. “ALKYLATING AGENT”
• Umumnya menimbulkan Alkilasi O
O6Me-G ~ A
O4Me-T ~ C
3. “BULKY AROMATIC AGENT”
• Bermacam=macam senyawa
• Umumnya terikat pada basa purin
• Karena bentuk molekul yagn besar  mengubah bentuk Helix
ganda DNA  mengganggu replikasi  DELESI
• Karena terikat pada basa purin  Hidrolisis  “Apurinic Site” 
“Random Base Pairing”  MUTASI TITIK

4. “INTERCALATING AGENT”
• Dapat “menyisip” di antara 2 pasangan basa 
menimbulkan kesalahan replikasi  INSERSI
CHEMICAL MUTAGENESIS
DEAMINATION (HNO , HSO ¯ )3 3

URACIL
CYTOSINE (ENOL) (C) (Keto)
(C)
Predominant tautomer of U *
(keto) ~ T  can pair with A

Hypoxanthine
(Enol) (HX) (Keto)
Predominant tautomer of Hx (keto)
~ G  T can pair with C

Sincer

Singer & Kus Hierek :

An. Rev. Biochemis 51 (1982), 655 --90
 CHEMICAL MUTAGENESIS
SHIFT OF PREFERED TAUTOMER
CYTOSINE ( C )
Prefered tautomer :
AMINO
Pairs with G
AMINO IMINO

METHOXYAMINE

N4-Methoxy-Cytosine
Prefered tautomer :
IMINO
Be haves like T
Pairs with A
IMINO
AMINO
GC  AT

Singer & Kusmierek

Ann. Rev.Biochem 51 (1992), 655 - 93
 CHEMICAL MUTAGENESIS
AMBIGUOS BEHAVIOR

CYTOSINE IMINO
AMINO PREFERED TAUTOMER :
AMINO
HIDROXYL AMINE

N4-OH CYTOSINE
AMINO BOTH TAUTOMER IMINO
COEXIST
ACTS LIKE T
ACTS LIKE C
CAN PAIR WITH A
UNCHANGED BASE
PAIRING
Singer & Kusmierek
Ann. Rev.Biochem 51 (1992), 655 - 93
ALKYLATING AGENTS
• Alkylation is the
transfer of an alkyl
group from one
molecule to another.

• In alkylation, methyl
or ethyl groups are
transferred to
reactive sites on the
bases and to
phosphates in DNA
backbone.

• Correlated with an
adition to the
oxygen at 6 position
of guanine and 4
position of thymine.
ALKYLATING AGENTS

• Alkylations do not lead

immediately to
mispairing  but, they
do make the bond
between sugar and base
more labile, or more apt
to break.

• When this break occurs,

 apurinic site  cannot
be replicated properly
unless it is first repaired.

• If it is not repaired
insert the wrong base
across from an apurinic
site  a mutation.
ALKYLATING AGENTS

• Alkylation can also

enhance the tendency of
a base to form the rare
(wrong) tautomer 
changes the base-pairing
properties  mutations.
Alkylation-induced
specific mispairing.
The alkylation (in this case,
EMS-generated ethylation)
of the O-6 position of
guanine and the O-4
position of thymine can lead
to direct mispairing with
thymine and guanine,
respectively.
BULKY AROMATIC AGENT
BULKY ADDUCT

REPLICATION HYDROLYSIS
A PURININC SITE

DELETION REPLICATION

REPLICATION

DELETION

POINT
MUTATION
RANDOM BASE PAIRING
 Intercalating agents
 Intercalating agents are compounds, such as
proflavin, acridine and ethidium, that can bind to
the major and minor grooves of DNA and cause
addition or deletion of bases during replication. They
may result in a frameshift mutation, which can alter
the codon reading frame and result in aberrant DNA
transcription and replication.
Base Analogs
 Mutagen kimia yang memiliki struktur
analog dengan basa nitrogen DNA
 UV RADIATION

 Hanya akan memberi akibat apabila pada

rantai DNA ditemukan 2 basa pirimidin yang
terletak berdampingan
TT atau CC
  dimer pirimidin (dua basa pirimidin saling
berikatan)
 Dimer timin  tautomer enol, berpasangan
dengan G
 Tautomer citosin  tautomer imino,
berpasangan dengan A
TAUTOMER
SITOSIN (C)
Tautomer
Imino C
Mirip T
Dapat berpasangan
dengan A

TIMIN (T)
Tautomer
Enol T
Mirip C
Dapat berpasangan
dengan G
UV RADIATION

ADJACENT
PYRIMIDINES

PYRIMIDINE
DIMER

1ST
REPLICATION

2ND
REPLICATION

AT  GC GC  AT
C* : IMINO TAUTOMER OF C
T* : ENOL TAUTOMER OF T
 IONIZING RADIATIONS X – RAYS
 - RAYS
1. IONIZING RADIATIONS CAN GIVE RISE TO FREE RADICALS BY HOMOLYTIC
CLEAVAGE OF WATER (H2O)
H2O  H● + ● OH
(H ATOM) (HYDROXYL RADICAL)
2. FREE RADICALS CAN DAMAGE N BASES (Pu & Py) RESULTING
IN : 1. HYDROXYLATION
2. RING OPENING
3. DAMAGED BASES CAN :
1. BLOCK REPLICATION
2. CAUSE MUTATIONS DIRECTLY OR INDIRECTLY DUE TO ERROR
PRONE DNA REPAIR
4. FREE RADICALS CAN ALSO CAUSE SINGLE OR DOUBLE STRAND
BREAKS WHICH IF EXTENSIVE CANNOT BE REPAIRED  CELL DEATH
5. FREE RADICALS CAN ALSO ATACK PUFA GIVING MDA AS ONE OF
ITS END PRODUCT MDA CAN REACT WITH PURINE BASES
GIVING END PRODUCTS THAT BLOCK DNA REPLICATION
DNA DAMAGE DUE TO FREE RADICALS

Timin menjadi THYMINE GLYCOL

mirip C, sehingga G (A)

Timin menjadi 5●OH METHYL URACIL

mirip C, sehingga G (A)

Guanin menjadi 8-OH GUANINE

mirip T, sehingga A (C)

Adenin menjadi 8-OH ADENINE

mirip C, sehingga G (T)

OXIDATION PRODUCTS CAUSING MUTATIONS

DNA DAMAGE BY FREE RADICALS
CAUSING DNA REPLICATION BLOCK

4,6 8 – NH2-5-FORMAMIDO PYRIMIDINE

“ FAPY – A “
RING OPENED A

2.6. DI– NH2-4-OXO-5-FORMIDO PYRIMIDINE

“ FAPY – G “
RING OPENED G

ADDITION PRODUCT OF MDA TO G

 DNA DAMAGE BY FREE RADICALS
CAUSING DNA REPLICATION BLOCK

 Radikal bebas juga dapat memotong

rantai DNA
 Satu rantai  mudah diperbaiki
 Dua rantai  perbaikan kurang sempurna 
akan disertai delesi, atau rantai DNA terpotong
pada banyak lokus  sel mati
 prinsip radioterapi dengan sinar gamma
(sel kanker mengalami replikasi sangat cepat 
saat sel membelah, dinding sel menghilang  tidak
terlindung dari paparan sinar gamma)
Sel normal yang replikasi cepat (sel ujung rambut,
eritrosit, sel membran usus)  terpengaruh