DNA DAMAGE AND

REPAIR
MAHAYU DEWI ARIANI

BAGIAN BIOLOGI
FAKULTAS KEDOKTERAN
UNIVERSITAS DIPONEGORO

DNA Damage
1000 - 1000000 molecular lesions/cell/day
impede a cell's ability to carry out its function
and appreciably increase the likelihood of
tumor formation.
physical abnormalities in the DNA, such as
single and double strand breaks
recognized by enzymes

DNA Damage vs
Mutation
Mutation:
change in the base sequence of the DNA.
cannot be recognized by enzymes
once the base change is present in both DNA
strands
cannot be repaired
DNA damages often cause errors of DNA
synthesis during replication or repair and these
errors are a major source of mutation.

Sources of damage
endogenous damage
attack by reactive oxygen species

exogenous damage
external agents such as

ultraviolet [UV 200-300nm] radiation from the sun

x-rays and gamma rays
hydrolysis or thermal disruption
plant toxins
human-made mutagenic chemicals
cancer chemotherapy and radiotherapy
viruses

How can DNA become

damaged?
All for of bases in the DNA (A,G,T,C) can be covalently

modified at various position.

Hydrolysis of purine-deoxyribose bond leading to AP-site.
Mismatched bases because of a failure of proofreading
during DNA replication
Polymerase error rate about 1 in 1044
Deamination of C to U leading to mismatch
Crosslinks
Covalent linkages can be formed between base (Dimer
formation)

Breaks in the backbone

Broken phosphodiester bonds. Chemicals/radiation

REPAIR MECHANISMS NECESSARY FOR SURVIVAL.

Types of DNA Damage

Summarised
G

ds DNA Break

Mismatch

C-U deamination

ss Break
Thymidine dimer

Covalent X-linking

AP site

Consequences of DNA
damage
DNA DAMAGE

DNA REPAIR MECHANISMS

SHORT-TERM CONSEQUENCES
PHYSIOLOGICAL
DYSFUNCTION
Decreased cellular
proliferation

CELL DEATH
Genomic
instability

Defective signalling
pathways

ABNORMAL GROWTH &

METABOLISM
Impaired protein/ gene
expression

LONG-TERM CONSEQUENCES
Ageing

Cancer

Disease

DNA Repair
a collection of processes by which a cell identifies
and corrects damage to the DNA molecules
that encode its genome

DNA Repair Mechanisms

Single strand damage
Excision repair

BER (Base Excision Repair)

NER (Nucleotide Excision Repair)

Mismatch repair
Double-strand breaks
Non-Homolog End Joining repair (NHEJ)
Homologous Recombination repair

BER (Base Excision Repair)

repairs damage to a single base caused by
oxidation, alkylation, hydrolysis, or deamination.
The damaged base is removed by a DNA
glycosylase.
The "missing tooth" is then recognised by an
enzyme called AP endonuclease.
The missing part is then resynthesized by a
DNA polymerase, and
a DNA ligase performs the final nick-sealing
step.

BER enzymes:

e.g. functional diversity I

Oxidized & ring opened purines:

e.g. 8-oxoG, fapyG

E.coli

S. cerevisiae

H. sapiens

MutM (fpg)

hOGG1

endo VIII (Nei)

yOGG1&2
_ _ _ _

AlkA (not oxoG)

_ _ _ _

hMPG

MutM/fpg: formamidopyrimidine glycosylase

endo VIII/Nei: endonuclease VIII
OGG: 8-oxoguanine DNA glycosylase
NEIL: Nei endonuclease VIII-like
MPG: N-methylpurine-DNA glycosylase
AlkA: 3-methyladenine DNA glycosylase II

hNEIL1

BER enzymes:

e.g. functional diversity II

Oxidized & ring opened pyrimidines: e.g. T-glycol, fapy
E.coli

S. cerevisiae

H. sapiens

endo III (Nth)

Ntg1&2
_ _ _ _ _
_ _ _ _ _
_ _ _ _ _

hNTHL1

endo VIII (Nei)

AlkA
_ _ _ _ _

endo III : endonucleases III

Ntg: endonuclease III-like glycosylase
NTHL1: Nth endonuclease III-like 1
TDG: thymine-DNA glycosylase

hNEIL1-3
_ _ _ _
hTDG

BER
Long patch

Short patch

Other BER Proteins

Removal of abasic sites
APE1

AP endonuclease

(HAP1, REF1)

Gap filling
PCNA

Proliferating cell nuclear antigen

DNA pol

DNA polymerase

Sealing the nick

XRCC1

X-ray cross-complementing protein & ligase

accessory factor

LIG3

DNA ligase

NER (Nucleotide Excision

Repair)
NER is implicated as a back-up system for BER
Recognizes bulky lesions that block DNA replication (i. e.
lesions produced by carcinogens
It recognize more varieties of damage
It removes larger segnents of DNA (10-100s bases)
different complexes initiate NER in transcribed & nontranscribed DNA
NER involves the dual incision of unwound DNA either
side of the lesion by a multisubunit ATP-dependent
nuclease
the resultant gap is filled & sealed by the action of various
components including DNA polymerases & ligases

Human NER
XPB

: 3' to 5' helicase

XPD

: 5' to 3' helicase

XPA

:Binds damaged DNA

after XPC or RNA pol II
:binds damaged DNA;
recruits other NER proteins
:Endonuclease (3' incision);
stabilizes full open complex

XPC
XPG
XPF

:Part of endonuclease
(5' incision)

ERCC1 :Part of endonuclease

(5' incision)
RPA
:Stabilizes open complex
(with XPA)

Rad1/10

Rad2 inS.
cerevisiae

Mismatch Repair (MMR)

MMR corrects errors of DNA replication and
recombination that result in mispaired (but undamaged)
nucleotides
Deals with correcting mismatch of the bases;that is
failures to maintain normal base pairing
Recognition of a mismatch requires several different
proteins including one encoded by MSH2
Cutting the mismatch also requires some proteins,
including one encoded by MLH1
Defects in MMR elevate spontaneous mutation rates 101000x
Defects in MMR underlie human predisposition to colon
and other cancers

Mismatched
bases.

3'
T

5'
T

5'

OH 3'

Polymerase III can 3'

repair mismatches.

5'
T

5'
T
1

G
OH

OH

3'

METHYLATION-DIRECTED MISMATCHED BASE REPAIR

Mismatch

Answer

1. Where a mismatch occurs, the

correct base is located on the
methylated strand: the incorrect base
occurs on the unmethylated strand.

2. Enzymes detect mismatch and nick

unmethylated strand.

3. DNA polymerase I excises

nucleotides on unmethylated strand.

4. DNA polymerase I fills in

gap in 5' 3' direction.

5. DNA ligase links new and

old nucleotides.

Repaired Mismatch

NHEJ
DNA Ligase IV, a specialized DNA
ligase that forms a complex with
the cofactor XRCC4, directly joins
the two ends
To guide accurate repair, NHEJ
relies on short homologous
sequences called microhomologies
important before the cell has
replicated its DNA
The end-joining pathway of ds
break repair is mutagenic, because
it removes several base pairs at
the break site.

Homologous
Recombination
requires the presence of an identical or nearly identical
sequence to be used as a template for repair of the
break.
This pathway allows a damaged chromosome to be
repaired using a sister chromatid (G2 after DNA
replication) or a homologous chromosome as a
template.
DSBs caused by the replication machinery attempting
to synthesize across a single-strand break or
unrepaired lesion are typically repaired by
recombination.

Homologous
recombination in
eukaryotes
1. Ds break
2. dsDNA activates ATM kinase, which
activates exo-nucleases that create ss 3
ends. In a reaction that depends on
BRCA 1 & 2, Rad51 coats the ss 3 ends.
3. Rad51 and friends pair the 3 end with
the sister chromatid.
4. DNA polymerase elongates.
5. Pairing of the new DNA bridges the
gap.
6. The gap is filled and ligated.

Thank
you..