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PERBAIKAN DNA

(DNA REPAIR SYSTEM)

Dr. Ema Qurnianingsih, dr., M.Si


• It is essential for all living organisms to warrant accurate
functioning and propagation of their genetic information
 but, the genome is constantly exposed to various
environmental and endogenous agents
 DNA lesions
• Environmental damage can be induced by several chemical
reactive species and physical agents.
• Endogenous damages occur spontaneously and
continuously even under normal physiologic conditions
through intrinsic instability of chemical bonds in DNA
structure.
• The biological consequences of these damages usually depend on the chemical
nature of the lesion.
• Most of these lesions affect the fidelity of DNA replication, which leads to
mutations.
Morita et al, journal of nucleic acid, 2010.
• Terdapat 5 mekanisme utama sistem atau jalur
perbaikan DNA:
• Direct reversal
• Base excision repair
• Nucleotide excision repair
• Mismatch repair
• Recombination repair
DIRECT REVERSAL
• UV-induced pyrimidine dimers and alkylation adducts can be
directly repaired by DNA photolyases and alkyl transferases,
respectively.
• These repair systems are not followed by incision or resynthesis
of DNA.
• Mekanisme perbaikan dimer pirimidin dengan aktivitas enzim
fotoliase terjadi pada prokariota dan sebagian besar eukariota,
namun sayangnya pada primata (manusia, gorila, simpanse,
orang utan)  enzim fotoliase tidak ada
• UV-induced pyrimidine
dimers, such as cyclobutane
pyrimidine dimers (CPDs)
and (6-4) photo products,
disturb DNA replication and
transcription.

DNA photolyase system : CPD


photolyases repair UV-induced
CPDs utilizing photon energy
from blue or near-UV light 
electron transfer to FAD 
reduced FAD (FADH)  The
FADH in the photolyase
donates an electron to the CPD
Gambar. Perbaikan DNA yang megalami lesi  breakage of the cyclobutane
(dimer pirimidin) melalui aktivitas DNA fotoliase bond
Morita et al, journal of nucleic acid, 2010.
In E. coli

Gambar. Reversal of O6-Alkylguanine-DNA.

O6-alkylguanine is one of the most harmful alkylation adducts and can induce
mutation and apoptosis. Almost all species possess mechanisms to repair this
adduct .
O6-alkylguanine-DNA alkyltransferase (AGT) accepts an alkyl group on a
cysteine residue at its active site  this alkylated AGT is inactive.
Morita et al, journal of nucleic acid, 2010.
• AlkB homologues are conserved in many organisms including humans and E. coli.
• AlkB requires α-ketoglutarate and Fe(II) as cofactors to repair N1-methyladenine or
N3-methylcytosine via an oxidative demethylation mechanism
• AlkB oxidizes the methyl group using nonheme Fe 2+, O2, and α-ketoglutarate to
restore undamaged bases with subsequent release of succinate, CO2, and
formaldehyde.

• Eight AlkB homologues are known in


humans  ALKBH1, ALKBH2, and
ALKBH3 have been identified as
repair enzymes, each of which has a
different substrate specificity 
ALKBH3 repairs lesions only in
ssDNA and RNA. ALKBH1 and
ALKBH2 can act only on DNA
• E. Coli AlkB can repair a lesion in
both single-stranded DNA (ssDNA),
dsDNA and RNA
Morita et al, journal of nucleic acid, 2010.
BASE EXCISION REPAIR
• DNA's bases  may be modified by deamination,
depurination or alkylation.
• DNA is altered and damaged by various endogenous
and exogenous reactions
• The lesions caused by endogenous and exogenous
reactive species can be repaired through the base
excision repair (BER) pathway
• The most frequently used DNA repair pathway in cells
Endogenous and exogenous reaction that alter DNA
– Endogenous :
• Deamination of cytosine, adenine and guanine produce
uracil, hypoxanthine, and xanthine, respectively.
• Depurination and depyrimidination result in the formation
of an apurinic/apyrimidinic site (AP site).
• Reactive oxygen species (ROSs) generate 2,6-diamino-4-
hydroxy-5-formamidopyrimidine (FaPyG); 4,6-diamino-5
formamidopyrimidine (FaPyA); Thymine glycol, etc.
• DNA replication errors also introduce lesions into the DNA.
For example, DNA polymerases sometimes incorporate
mismatched bases or damaged nucleotides (such as dUMP
and 8-oxo-dGMP)
– Exogenous : DNA is susceptible to damage by agents such as
UV radiation, ionizing radiation (give raise to free radicals),
alkylating compounds, bulky aromatic agents.
• General mechanism
of BER : GLYCOSILASE
1. DNA glycosylase
 recognize the AP
site and remove its
AP ENDONUCLEASE
base
2. AP endonuclease
 removes the AP DEOXY RIBOSE
site and neighboring PHOSPHATAS
nucleotides
DNA POLYMERASE
3. The gap  filled
by DNA polymerase I DNA LIGASE
and DNA ligase.
NUCLEOTIDE EXCISION REPAIR
• In E. coli, proteins UvrA, UvrB, and
UvrC  involved in removing the
damaged nucleotides the dimer
induced by UV light

• The gap  filled by DNA


polymerase I and DNA ligase.

• In yeast, the proteins similar to


Uvr's are named RADxx ("RAD"
stands for "radiation"), such as
RAD3, RAD10. etc.
A schematic representation of models for the nucleotide excision repair
pathway controlled by Uvr proteins.
Morita et al, journal of nucleic acid, 2010.
DNA REPAIR (2)
OLIGONUCLEOTIDE EXCISION
DINUCLEOTIDE ADDUCT

EXCINUCLEASE

DNA POLYEMRASE
+ DNA LIGASE

SANCAR.
ANN. REY. BIOCHEM. 1986
57. 29 - 47
MISS-MATCH REPAIR (MMR)
• DNA damage, if unrepaired, has the potential to generate
mutations in somatic or germline cells, which can alter cellular
phenotype and cause dysfunction and disease
• MMR corrects DNA mismatches generated during DNA
replication, thereby preventing mutations from becoming
permanent in dividing cells
• Mutations in the genes involved in MMR are associated with
increased predisposition to human hereditary nonpolyposis
colorectal cancers
• The prototypical Escherichia coli MMR pathway has been
extensively studied and is well characterized both biochemically
and genetically. Thus, E. coli MMR is a useful and important
framework for understanding eukaryotic MMR
• To repair mismatched bases, the system has to know which
base is the correct one., but no methyl
• Immediately after DNA replication, the template strand has been
methylated, but the newly synthesized strand is not methylated
yet.
• MMR in E. coli involves three steps : recognition, excision and
resynthesis
A Schematic representation of missmatch repair in E. coli
Guillotin and Martin, Experimental cell research, 2014
• MutS recognizes
base-base
mismatches and
small nucleotide
insertion/deletion (ID)
mispairs,  the
“mismatch
recognition” protein 
it forms a sliding
clamp able to
translocate in either
direction on the DNA
until it recognizes a
mismatch

• This recognition alters


the MutS
MutL, another MMR-
associated
heteroduplex is
composed of MLH1,
with either PMS2
(MutLα), PMS1
(MutLβ) or MLH3
(MutLγ). MutLα is
the most prominent
MutL complex and
when complexed
with MutS, acts as
an endonuclease
• MutL is able to
recognize and
excise the lagging
strand from the
mismatch.

• Exonuclease 1
(Exo1) is recruited to
excise nucleotides in
a 5′->3′ direction. In
the case of 3′
excision, Exo1
recruits replication
factor C (RFC) to
• Replication protein A
(RPA), a binding-
factor, stabilizes the
single-stranded DNA
and inhibits Exo1 to
prevent further
degradation.

• After removing the


mismatch, the final
step is to
resynthesize the
excised-DNA by
DNA polymerase δ
and to ligate the
remaining nick by
DNA REPAIR (3)
RECOMBINATIONAL REPAIR
DINUCLEOTIDE ADDUCT

REPLICATION
DNA POLYMERASE

RECOMBINASE

DNA POLYMERASE

RECOMBINASE

RESOLVASE + DNA LIGASE


SANCAR.
ANN. REY. BIOCHEM. 1986
57. 29 - 47
DNA REPAIR ( 4 )
CROSS LINK REPAIR
INTERSTRAND CROSSLINK

EXCINUCLEASE

RECOMBINATIONAL
REPAIR
Karena celah yang terbentuk
cukup lebar, maka tidak bisa
lewat eksisi oligonukleotida
EXCINUCLEASE

DNA POLYMERASE
+ DNA LIGASE
SANCAR.
ANN. REY. BIOCHEM. 1986
57. 29 - 47
DOUBLE STRANDED BREAK REPAIR (DSBR)

KU

• Bila hanya satu


DNA PK
rantai DNA yang
terputus  DNA
repair dengan DNA
ligase  mudah
• Dua rantai DNA
putus (patahan dua Exonuclease
rantai / PDR)  (extra single strand destroyed)
GAP
perbaikan PDR atau
DSBR)  rumit GAP
DNA Ligase
(GAP Closed)

DOUBLE STRANDED
BREAK REPAIRED
• Terdapat dua mekanisme DSBR
– Homologous recombination (HR)
• Membutuhkan template DNA yang utuh untuk
menyambungkan ujung DNA dan mengkopi sekuens
yang hilang dari area sekitar DSB
• Merupakan jalur utama pada prokariota dan eukariota
tingkat rendah
– Nonhomologous end-joining (NHEJ)
• Jalur ini menggunakan sedikit atau tidak sama sekali
homologi untuk menyambung ujung DNA
• Error prone
• Pada eukariota tingkat tinggi termasuk manusia
Nonhomologous end-joining (NHEJ)
General outline of NHEJ
A. Jalur klasik :
NHEJ dimulai dengan rekognisi ujung DNA pada
DSB oleh Ku70/80  menarik DNA-PKcs 
autofosforilasi  perubahan konformasi pada
kompleks ikatan DNA-Ku70/80 & DNA-PKcs 
jika ujung kompatibel, maka ligase IV (komplek
dengan XRCC4) melakukan ligasi DNA
A. Jalur alternatif :
– tidak tergantung Ku70/80, ligase IV dan XRCC4
– Menggunakan regio mikrohomologi untuk
menghubungkan ujung DNA yang terpotong
sebelum penggabungan rantai DNA
– Enzim yang terlibat masih belum jelas
DOUBLE STRANDED BREAK REPAIR (DSBR)
DOUBLE STRANDED BREAK REPAIR (DSBR)