DNA REPAIR

Dr. Farina Hanif Ph.D.; Postdoc

Assistant Professor, Molecular Pathology
Why DNA Repair is Important???

The End!
 DNA REPAIR
DNA is the only biological molecule that
is repaired
DNA damage
Alteration to the chemical structure of
DNA
Mutation
Change in the sequence of DNA
NATURE OF DNA DAMAGE

 Loss of bases
 Modification of bases
 Inter/intra-strand crosslinks
 DNA strand breakage (single and
double strand)
 CAUSES OF DNA DAMAGE
 Endogenous factors
Spontaneous
- Errors in proofreading
- Deamination of bases
- Depurination/Depyrimidination
Induced
- Byproducts of normal cellular processes
(reactive oxygen species etc )
 Exogenous factors
- UV irradiation (sunlight)
- High energy irradiation (x-rays)
- Mutagenic chemicals (Mustard gas, cigarette
smoke, food additives)
 ERRORS IN PROOFREADING

Incorporation of the wrong base/s

resulting in mismatches

Approximate error rate = 10-9

 Accuracy of Replication

 1000 bases/second =
lots of typos!
 Replication = 10-3

 Proofreading= 10-6
 3’-5’ exonuclease activity

 Repair System = 10-9

  DEAMINATION

May be spontaneous or induced by

chemicals
Cytosine  Uracil
Adenine  Hypoxanthine
Guanine  Xanthine
Thymine  ??
  DEAMINATION

• Deamination leads to unusual base pairing in DNA

- Uracil pairs with adenine

- Hypoxanthine pairs with cytosine
FAILURE TO REPAIR A DEAMINATED
BASE = A POINT MUTATION
Parental strand

C T U T C
Deamination Mutation
GA A AG
New strand

C T U
C T C DNA
Replication
GAGAG
New strand
C T C T C
Unchanged
GAGAG
Parental strand
  DEPURINATION/
DEPYRIMIDINATION
• Cleavage of the glycosidic bond removes
bases
C T C T C

GAG G

– Abasic (Apurinic/apyrimidinic, AP sites)

– ~2000-10,000 purines lost per mammalian
cell/24 hr
FAILURE TO REPAIR ABASIC
SITES = DELETIONS
Parental strand
C T C T C
Unchanged
GAGAG
New strand
C T C T C DNA
Replication
GAG G New strand

C T C C
AP site Mutation
GAG G
Parental strand
 REACTIVE OXYGEN SPECIES
Generated during normal aerobic respiration
– Superoxides, O2-,
.
– Hydroxyl ions (OH )
– H2O2

Most biological damage by OH.

Guanine 8-oxodG
 Exogenous – UV IRRADIATION

Dimerizes adjacent
thymine residues.
The dimer creates a
kink in the DNA that
blocks the
progression DNA
polymerase
 HIGH-ENERGY RADIATION
X-rays and gamma rays may directly
break DNA strands

and/or generate reactive oxygen species

 Exogenous – CHEMICALS
• Alkylating agents (e.g., mustard gas)
– Add CH3/CH2CH3 groups to N and O groups of
bases.
– O6 of guanine particularly susceptible.
6-ethyl guanine acts as an analogue of adenine
and pairs with thymine.
• Polycyclic Hydrocarbons (cigarette smoke,
exhaust fumes etc)
 Exogenous – CHEMICALS
• Food Additives
– Nitrates and Nitrites
– Metabolized to Nitronium ion/Nitrous acid
• Chemotherapeutic drugs
– Base Analogues (e.g. 5-bromouracil, 5BU,
can pair with adenine or guanine)
• Intercalating Agents
– Acridine dyes (e.g., proflavin)
– Interfere with DNA replication
☺ METHODS OF REPAIR
☺ Excision repair
- Base excision
- Nucleotide excision
☺ Mismatch repair
☺ Direct repair
☺ Recombination repair
EXCISION REPAIR

Recognition of damage

Removal of damage

Resynthesis of gap

Ligation
 EXCISION REPAIR
Two types of excision repairs
• Base Excision Repair
Repair of methylated, deaminated, oxidized
bases and AP sites.

• Nucleotide Excision Repair

Repair of large adducts or distortion in the
double helical structure of DNA (pyrimidine
dimers, benzo(a)pyrene)
 BASE EXCISION REPAIR

Glycosylase

AP Lyase
endonuclease

DNA polymerase

DNA ligase
 NUCLEOTIDE EXCISION REPAIR
Thymine dimer

UvrAB excinuclease
A A
B

UvrC excinuclease
A A
B HvrD Helicase

DNA polymerase

DNA ligase

No thymine dimer
NER ASSOCIATED DISEASES
• Xeroderma pigmentosum
• Cockayne Syndrome
• PIBIDS (photosensitivity, ichthyosis, brittle
hair, impaired intelligence, decreased
fertility, short stature)

- Characterized by an increased sensitivity

to sunlight
XERODERMA PIGMENTOSUM
• Can be caused by defects in any one of
seven different NER genes
– Predisposition to skin cancer
– Pigmentation abnormalities
– Premalignant lesions
– Degeneration of the
nervous system
 ☺ EXCISION REPAIR
• Base excision repair • Nucleotide excision repair
– Repair of modified – Repair of adducts and large
bases distortions in DNA double
– Glycosylase removes helix
base, leaves backbone – Double excision removes
intact damage as an
– AP endonuclease cut oligonucleotide (12-13 nt
backbone, AP lyase in E. Coli, 27-29 nt in
removes sugar humans)

DNA polymerase fills gap

DNA ligase seals nick
The forms of DNA damage mentioned so far,
the repair machinery can easily tell which
base(s) have been damaged.
Cytosine deamination gives rise to Uracil,
which shouldn’t be present in DNA
Similarly bases modified by adducts are
recognizable
as wrong.

What happens when the cell can’t easily

distinguish which base is wrong.
 ☺ Mismatch repair

• Repair of replication (proofreading)

errors

• Recognition of bases that do not form

normal Watson-Crick pairs
 ☺ Mismatch repair
• How do the repair enzymes recognize
which strand to fix???
CH3 CH3
?
AGA T C T C T T CGA T C
x
T C T AGAG C AGC T AG
?
 ☺ Mismatch repair
CH3 CH3

MutS/MutL

MutH CH3 CH3

DNA Polymerase
CH3 CH3

DNA Ligase
CH3 CH3
 HEREDITARY NONPOLYPOSIS
COLORECTAL CANCER (HNPCC)

• Lynch syndrome
• Accounts for 2 -10% of all colon
cancers
• Caused by defects in mismatch repair
genes.
☺ Direct repair
DNA STRAND BREAKS
 DNA STRAND BREAKS
• 10 -100 naturally occurring double-strand
breaks per cell per day

• Two mechanisms for repair

– Homologous recombination repair (HRR)
– Nonhomologous recombination repair
(NHRR)
Homologous recombination repair
(HRR)
Nonhomologous recombination
repair (NHRR)
 HRR Vs. NHRR
HRR NHRR
• Identical copies • Small deletions
made occur

• Only possible in the • Any time in the

S and G2 phase of cell cycle
the cell cycle
LexA, RecA in the SOS
response
Summary!!!