dna repair. dna is the only biological molecule that is repaired dna damage alteration to the...
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DNA REPAIRDNA REPAIR
DNA REPAIRDNA 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 NATURE OF DNA DAMAGEDAMAGE
Loss of bases Modification of bases Inter/intra-strand crosslinks DNA strand breakage (single and
double strand)
CAUSES OF DNA DAMAGECAUSES 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)
ERRORSERRORS IN PROOFREADING IN PROOFREADING
Incorporation of the wrong base/s resulting in mismatches
Approximate error rate = 10-9
DEAMINATION DEAMINATION
May be spontaneous or induced by chemicals
Cytosine Adenine Guanine Thymine
Uracil
Hypoxanthine
Xanthine
??
DEAMINATION DEAMINATION
• Deamination leads to unusual base pairing in DNA
- Uracil pairs with adenine
- Hypoxanthine pairs with cytosine
FAILURE TO REPAIR A DEAMINATED FAILURE TO REPAIR A DEAMINATED BASE = A POINT MUTATIONBASE = A POINT MUTATION
T C T C
A G A G
C
G
Deamination
DNA
Replication
T C T CCUnchanged
A G A GGParental strand
New strand
A A A GGMutation
T U T CC
Parental strand
New strand
U
DEPURINATION/DEPURINATION/DEPYRIMIDINATIONDEPYRIMIDINATION
• Cleavage of the glycosidic bond removes bases
– Abasic (Apurinic/apyrimidinic, AP sites)– ~2000-10,000 purines lost per mammalian
cell/24 hr
CT T C
A G G
C
G
FAILURE TO REPAIR ABASIC SITES FAILURE TO REPAIR ABASIC SITES = DELETIONS= DELETIONS
CT T C
A G G
C
G
DNA
Replication
Unchanged
Mutation
A G A GGNew strand
CT CC
New strand
T C T CC
Parental strand
GA GGParental strand
AP site
REACTIVE OXYGEN SPECIESREACTIVE OXYGEN SPECIES
Generated during normal aerobic respiration
– Superoxides, O2-,
– Hydroxyl ions (OH.)
– H2O2
Most biological damage by OH.
Guanine 8-oxodG
Exogenous – UV IRRADIATIONExogenous – UV IRRADIATION
Dimerizes adjacent thymine residues.
The dimer creates a kink in the DNA that blocks theprogression DNA polymerase
HIGH-ENERGY RADIATIONHIGH-ENERGY RADIATION
X-rays and gamma rays may directly break DNA strands
and/or generate reactive oxygen species
• 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 – CHEMICALSExogenous – CHEMICALS
Exogenous – CHEMICALSExogenous – 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 CYCLETHE CELL CYCLE
☺☺ METHODS OF REPAIRMETHODS OF REPAIR
☺☺ Excision repair
- - Base excision
-- Nucleotide excision
☺☺ Mismatch repair
☺☺ Recombination repair
EXCISION REPAIREXCISION REPAIR
Removal of damage
Resynthesis of gap
Ligation
Recognition of damage
EXCISION REPAIREXCISION 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 REPAIRBASE EXCISION REPAIR
Glycosylase
AP endonuclease
DNA polymerase
DNA ligase
AP Lyase
NUCLEOTIDE EXCISION REPAIRNUCLEOTIDE EXCISION REPAIR
DNA polymerase
DNA ligase
urvAB excinuclease
BA A
BA A
urvC excinuclease
Thymine dimer
No thymine dimer
NER ASSOCIATED DISEASESNER 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 12A 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 PIGMENTOSUMXERODERMA 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
• Base excision repair– Repair of modified
bases– Glycosylase removes
base, leaves backbone intact
– AP endonuclease cut backbone, AP lyase removes sugar
• Nucleotide excision repair– 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)
☺ EXCISION REPAIREXCISION REPAIR
DNA polymerase fills gapDNA 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???
?
?
CH3CH3
A
T
G A T C T C
C T A G A G
T
C
T C G A T C
A G C T A G
x
☺ Mismatch repair
CH3 CH3
CH3 CH3
DNA Polymerase
DNA Ligase
CH3 CH3
CH3 CH3
MutS/MutL
MutH
HEREDITARY NONPOLYPOSIS HEREDITARY NONPOLYPOSIS COLORECTAL CANCER (HNPCC)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 BREAKSDNA STRAND BREAKS
DNA STRAND BREAKSDNA 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 Homologous recombination repair (HRR)repair (HRR)
Nonhomologous recombination Nonhomologous recombination repair (NHRR)repair (NHRR)
HRR Vs. NHRRHRR Vs. NHRR
HRR
• Identical copies made
• Only possible in the S and G2 phase of the cell cycle
NHRR
• Small deletions occur
• Any time in the cell cycle
Defects in DNA-repair systems associated Defects in DNA-repair systems associated with certain cancerswith certain cancers
The End!The End!