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.
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
• 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 perform a 3′->5′ excision.
• Replication protein A
(RPA), a binding-factor,
stabilizes the single-
stranded DNA and inhibits
Exo1 to prevent further
degradation.
REPLICATION
DNA POLYMERASE
RECOMBINASE
DNA POLYMERASE
RECOMBINASE
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
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)