DNA REPAIR

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DNA REPAIR
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
 DEAMINATION
May be spontaneous or induced by
chemicals
Cytosine
Adenine
Guanine
Thymine




Uracil
Hypoxanthine
Xanthine
??
 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
Mutation
Deamination
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)
• Intercalating Agents
– Acridine dyes (e.g., proflavin)
– Interfere with DNA replication
THE CELL CYCLE
☺ METHODS OF REPAIR
☺ Excision repair
- Base excision
- Nucleotide excision
☺ Mismatch 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
urvAB excinuclease
A
B
A
urvC excinuclease
A
B
A
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
Vignette 12
A 3-year-old boy, was referred to the dermatology clinic for evaluation
of severe sun sensitivity and freckling.
On physical examination, he was photophobic and had conjunctivitis
and prominent freckled hyperpigmentation in sun-exposed areas; his
development and physical examination were otherwise normal.
The parents of the child revealed that they were first cousins; no one
else in the family was similarly affected.
The dermatologist explained that the boy had classic features of
xeroderma pigmentosum (XP), that is, "parchment-like pigmented skin".
To confirm the diagnosis, he had a skin biopsy to evaluate DNA repair
and ultraviolet (UV) radiation sensitivity in his skin fibroblasts.
The results of this testing confirmed the diagnosis of XP. Despite
appropriate preventive measures, the boy developed metastatic
melanoma at 15 years of age and died 2 years later.
His parents had two other children; neither was affected with XP.
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
bases
– Glycosylase removes
base, leaves backbone
intact
– AP endonuclease cut
backbone, AP lyase
removes sugar
– Repair of adducts and large
distortions in DNA double
helix
– Double excision removes
damage as an
oligonucleotide (12-13 nt
in E. Coli, 27-29 nt in
humans)
DNA polymerase fills gap
DNA ligase seals nick
☺ 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
CH3
CH3
MutS/MutL
MutH
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 MSH2, MSH6, MLH1, PMS1 or
PMS2
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
• Identical copies
made
NHRR
• Small deletions
occur
• Only possible in the • Any time in the
S and G2 phase of
cell cycle
the cell cycle
Defects in DNA-repair systems associated
with certain cancers
The End!
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