DNA Damage & Repair

DNA damage, repair & mutation

mutagens
DNA damage Normal DNA

DNA repairing systems

DNA repair DNA mutation

In
fertile
Cell Defect of
death metabolism Defect of Cell evolution
growth regulation polymorphism Inborn
error
Cancer
 Mutagen Kimiawi :
Ni
Be
Asbes (kanker paru)
Bahan pengalkil : vynil khlorida
mustard nitrogen
Nitrosurea & nitrosamin
Zat warna azo
Hidrokarbon polisiklik
Berbagai produk mikroba
Berbagai produk jamur
Hormon dietilstilbestrol (Kanker vagina & testis)
Naftilamin- (kanker kandung kencing)
 Mutagen fisikal

Sinar-X

Sinar -

Sinar ultraviolet dg pj glb 320 nm

 Mutagen biologi

Virus DNA :
SV40, Virus polioma,
Adenovirus, Virus Herpes
terbukti mampu menimbulkan tumor pada binatang coba dan biakan
sel,

Virus RNA : Retrovirus RNA utas tunggal

terbukti terhadap biakan sel.
 Kerusakan akibat kimiawi

Depurinasi  apurinic = AP sites

Metilasi basa
Deaminasi basa : C  U ; A  HX; G  X
Depurination
Deamination of bases
 Fisikal :
Thymime dimer
Effect of UV light
Thymine dimer
Mutation
Via
deaminat
ion
DNA repairing Enzymes

1.DNA glycosylase
2.AP endonuclease
3.DNA polymerase III
4.Exonuclease
DNA Repair
 Table 5-2. Inherited Syndromes with Defects in DNA
Repair
NAME
MSH2, 3, 6, MLH1, PMS2
Xeroderma pigmentosum
(XP) groups A -G
XP variant
Ataxia -telangiectasia (AT)
BRCA-2
Werner syndrome
Bloom syndrome
Fanconi anemia groups A -G
46 BR patient
PHENOTYPE
colon cancer
skin cancer, cellular UV sensitivity,
neurological abnormalities
cellular UV sensitivity
leukemia, lymphoma, cellular g-ray
sensitivity, genome instability
breast and ovarian cancer
premature aging, cancer at several
sites, genome instability
cancer at several sites, stunted
growth, genome instability
congenital abnormalities, leukemia,
genome instability
hypersensitivity to DNA-damaging
agents, genome instability
ENZYME OR PROCESS
AFFECTED
mismatch repair
nucleotide excision-repair
translesion synthesis by DNA
polymerase d
ATM protein, a protein kinase
activated by double-strand breaks
repair by homologous recombination
-exonuclease and DNA helicase
accessory DNA helicase for
replication
DNA interstrand cross-link repair
DNA ligase I
 Without DNA Repair,
Spontaneous DNA Damage
Would Rapidly Change DNA
Sequences
 Figure 5-46. A summary of spontaneous alterations
likely to require DNA repair.

The sites on each nucleotide that are known to be modified by spontaneous oxidative
damage (red arrows), hydrolytic attack (blue arrows), and uncontrolled methylation by
the methyl group donor S-adenosylmethionine (green arrows) are shown, with the width
of each arrow indicating the relative frequency of each event.
(After T. Lindahl, Nature 362:709715, 1993. © Macmillan Magazines Ltd.)
 Figure 5-47. Depurination and deamination.

These two reactions are the most frequent spontaneous chemical reactions known to create serious DNA
damage in cells. Depurination can release guanine (shown here), as well as adenine, from DNA. The major
type of deamination reaction (shown here) converts cytosine to an altered DNA base, uracil, but
deamination occurs on other bases as well. These reactions take place on double-helical DNA; for
convenience, only one strand is shown.
Figure 5-48. The thymine dimer.

This type of damage is introduced into

DNA in cells that are exposed to
ultraviolet irradiation (as in sunlight).
A similar dimer will form between any
two neighboring pyrimidine bases (C or
T residues) in DNA.
 Figure 5-49. How chemical modifications of nucleotides
produce mutations.
(A) Deamination of cytosine, if uncorrected,
results in the substitution of one base for
another when the DNA is replicated. As
shown in Figure 5-47, deamination of
cytosine produces uracil. Uracil differs from
cytosine in its base-pairing properties and
preferentially base-pairs with adenine. The
DNA replication machinery therefore adds an
adenine when it encounters a uracil on the
template strand.
(B) Depurination, if uncorrected, can lead to either
the substitution or the loss of a nucleotide pair. When
the replication machinery encounters a missing
purine on the template strand, it may skip to the next
complete nucleotide as illustrated here, thus
producing a nucleotide deletion in the newly
synthesized strand. Many other types of DNA
damage (see Figure 5-46) also produce mutations
when the DNA is replicated if left uncorrected.
The DNA Double Helix Is Readily
Repaired
DNA Damage Can Be Removed
by More Than One Pathway
Figure 5-50 A. A comparison of two major DNA repair
pathways; Base excision repair

(A) Base excision repair. This pathway starts with a

DNA glycosylase. Here the enzyme uracil DNA
glycosylase removes an accidentally deaminated
cytosine in DNA.
After the action of this glycosylase (or another DNA
glycosylase that recognizes a different kind of
damage), the sugar phosphate with the missing
base is cut out by the sequential action of AP
endonuclease and a phosphodiesterase. (These
same enzymes begin the repair of depurinated
sites directly.)
The gap of a single nucleotide is then filled by DNA
polymerase and DNA ligase. The net result is that
the U that was created by accidental deamination is
restored to a C.
The AP endonuclease derives its name from the
fact that it recognizes any site in the DNA helix that
contains a deoxyribose sugar with a missing base;
such sites can arise either by the loss of a purine
(apurinic sites) or by the loss of a pyrimidine
(apyrimidinic sites).
Figure 5-50 B. A comparison of two major DNA repair
pathways; Nucleotide excision repair

(B) Nucleotide excision repair.

After a multienzyme complex has
recognized a bulky lesion such as a
pyrimidine dimer (see Figure 5-48),
one cut is made on each side of the
lesion, and an associated DNA
helicase then removes the entire
portion of the damaged strand.
The multienzyme complex in
bacteria leaves the gap of 12
nucleotides shown; the gap
produced in human DNA is more
than twice this size.
The nucleotide excision repair
machinery can recognize and repair
many different types of DNA
damage.
Figure 5-51. The recognition of an unusual nucleotide
in DNA by base-flipping.

The DNA glycosylase

family of enzymes
recognizes specific
bases in the
conformation shown.
Each of these enzymes
cleaves the glycosyl
bond that connects a
particular recognized
base (yellow) to the
backbone sugar,
removing it from the
DNA.

(A) Stick model

(B) space-filling model.
The Chemistry of the DNA Bases
Facilitates Damage Detection
 Figure 5-52. The
deamination of DNA
nucleotides.
In each case the oxygen atom that is added in
this reaction with water is colored red.
(A) The spontaneous deamination products of
A and G are recognizable as unnatural when
they occur in DNA and thus are readily
recognized and repaired.
The deamination of C to U was previously
illustrated in Figure 5-47; T has no amino
group to deaminate.
(B) About 3% of the C nucleotides in vertebrate
DNAs are methylated to help in controlling
gene expression (discussed in Chapter 7).
When these 5-methyl C nucleotides are
accidentally deaminated, they form the natural
nucleotide T.
This T would be paired with a G on the
opposite strand, forming a mismatched base
pair.
Double-Strand Breaks are
Efficiently Repaired
Figure 5-53. Two different types of end-joining for repairing
double-strand breaks.

(A) Nonhomologous
end-joining alters the
original DNA
sequence when
repairing broken
chromosomes. These
alterations can be
either deletions (as
shown) or short
insertions.

(B) Homologous end-

joining is more
difficult to
accomplish, but is
much more precise.
Cells Can Produce DNA Repair
Enzymes in Response to DNA
Damage
DNA Damage Delays Progression
of the Cell Cycle
Exonucleolytic proofreading by DNA
polymerase during DNA replication.

In this
example, the
mismatch is
due to the
incorporation of
a rare,
transient
tautomeric
form of C,
indicated by an
asterisk.
But the same
proofreading
mechanism
applies to any
misincorporatio
n at the
growing 3-OH
end
 DNA glycosylase
At least 8 DNA glycosylases in human.
UNG = uracyl DNA glycosylase remove U from DNA
Depurination
Apurinic Endonuclease
 Excision repair
Basic steps : recognize damage
Remove damage by excising part of one strand
Resynthesize to fill gap; genetic information from other strand used
Ligate to restore continuity of DNA

Base excision repair:

Nucleotide excision repair :
Glycosylase remove base, leaves
Double excision removes
backbone intack
oligonucleotide (12-13 nt in Ecoli, 27-
AP endonuclease cuts backsbone, 29 nt in humans)
DNA polymerase fills the gap
AP lyase removes sugar
DNA ligase seals the nick
DNA polymerase fills the gap
DNA ligase seals the nick
 Base excision repair
Repairs methylaed, deaminated, oxidized bases, and abasic site

The damaged site is recognised by DNA glycosylase

The base is removed by cleavage of glycosyl bound that connects it
deoxyribose. The sugar phosphate backbone is not-broken. This
leaves a AP site.
The sugar-phosphate backbone is cleavage 5’ to the abasic site by an
AP endonuclease.
There is also a cleavage at the 3’ side of this site to remove the sugar
residue; this can be done by some AP endonucleases or an AP lyase
activity.
Single nucleotide gap is filled by a DNA polymerase.
Remaining nick is sealed by a DNA ligase
 Nucleotide excision repair
Repair pirimidin dimers, large adduct or distorsion of the dsDNA. Chemical adduct
with carcinogen aflatoxin, chemotherapeutic cisplatin, mismatched bases, small
loops in DNA.

The damage base is recognized by a DNA complex.

The segment bound the damage is excised by an enzyme complex that
makes two nicks in the same strand, one on either side of the damage.
An oligonucleotide (27 -29 nt in humansw, 11-13 nt in E coli,) is
released.
Resynthesis; a DNA polymerase fills the gap, using the oposite strand
as a template.
DNA ligase seals the remaining nick.
DNA repair of thymin dimer
 Enzymatic demethylation
Alkylating bases are mutagenic.
Methyl guanine basepair with T rather than C. Replication this basepair
lead to mutation.
Glycosylases recognizes the the methylated bases, trigger base excision
repair.
Cell also has special protein that recognize methyl guanine in DNA, and
directly remove the methyl group by enzyme methyl guanine methyl
transsferase (MGMT), transfer it to cystein in their protein, make the
protein inactive and degraded through the ubiquitin proteolytic pathway.
 Photoreactivation
Repair cyclobutane pyrimidine dimers,

Photolyase bind to the damage.

Upon absorbing light, it catalyse reversal of the bonds
between adjacent pyrimidins,
Not significant in mammals
Cell responses to DNA damage
Apoptosi
s in DNA
damage
Mutation
Frameshift mutation
Frameshift mutation
Segregation of mutant
 Table 5-2. Inherited Syndromes with Defects in DNA
Repair
NAME
MSH2, 3, 6, MLH1, PMS2
Xeroderma pigmentosum
(XP) groups A -G
XP variant
Ataxia -telangiectasia (AT)
BRCA-2
Werner syndrome
Bloom syndrome
Fanconi anemia groups A -G
46 BR patient
PHENOTYPE
colon cancer
skin cancer, cellular UV sensitivity,
neurological abnormalities
cellular UV sensitivity
leukemia, lymphoma, cellular g-ray
sensitivity, genome instability
breast and ovarian cancer
premature aging, cancer at several
sites, genome instability
cancer at several sites, stunted
growth, genome instability
congenital abnormalities, leukemia,
genome instability
hypersensitivity to DNA-damaging
agents, genome instability
ENZYME OR PROCESS
AFFECTED
mismatch repair
nucleotide excision-repair
translesion synthesis by DNA
polymerase d
ATM protein, a protein kinase
activated by double-strand breaks
repair by homologous recombination
-exonuclease and DNA helicase
accessory DNA helicase for
replication
DNA interstrand cross-link repair
DNA ligase I