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!