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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. DNA REPAIR. NATURE OF DNA DAMAGE. Loss of bases Modification of bases Inter/intra-strand crosslinks

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dna repair1
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

DNA REPAIR
slide3

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
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
 ERRORS 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

??

deamination1
 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

G

A

A

A

G

DNA

Replication

C

T

C

T

C

G

A

G

A

G

Parental strand

FAILURE TO REPAIR A DEAMINATED BASE = A POINT MUTATION

Mutation

Deamination

New strand

C

T

U

C

T

C

G

A

G

A

G

New strand

Unchanged

depurination depyrimidination

C

C

T

T

C

G

A

G

G

 DEPURINATION/DEPYRIMIDINATION
  • Cleavage of the glycosidic bond removes bases
    • Abasic (Apurinic/apyrimidinic, AP sites)
    • ~2000-10,000 purines lost per mammalian cell/24 hr
slide10

FAILURE TO REPAIR ABASIC SITES = DELETIONS

Parental strand

C

T

C

T

C

G

A

G

A

G

New strand

C

C

T

T

C

DNA

Replication

G

A

G

G

New strand

C

T

C

C

G

G

A

G

Parental strand

Unchanged

AP site

Mutation

reactive oxygen species
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
 Exogenous – UV IRRADIATION

Dimerizes adjacent thymine residues.

The dimer creates a kink in the DNA that blocks the

progression DNA polymerase

high energy radiation
 HIGH-ENERGY RADIATION

X-rays and gamma rays may directly break DNA strands

and/or generate reactive oxygen species

slide14

 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)
slide15

 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
methods of repair
☺ METHODS OF REPAIR

☺ Excision repair

- Base excision

-Nucleotide excision

☺ Mismatch repair

☺ Recombination repair

excision repair
EXCISION REPAIR

Recognition of damage

Removal of damage

Resynthesis of gap

Ligation

excision repair1
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
BASE EXCISION REPAIR

Glycosylase

AP endonuclease

AP Lyase

DNA polymerase

DNA ligase

slide21

NUCLEOTIDE EXCISION REPAIR

A

A

B

Thymine dimer

urvAB excinuclease

urvC excinuclease

A

A

B

DNA polymerase

DNA ligase

No thymine dimer

ner associated diseases
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

slide23

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
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

slide25
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 REPAIR

  • DNA polymerase fills gap
  • DNA ligase seals nick
mismatch repair
☺ Mismatch repair
  • Repair of replication (proofreading) errors
  • Recognition of bases that do not form normal Watson-Crick pairs
slide27

☺ Mismatch repair

  • How do the repair enzymes recognize which strand to fix???

?

CH3

CH3

A

G

A

T

C

T

C

T

T

C

G

A

T

C

x

T

C

T

A

G

A

G

C

A

G

C

T

A

G

?

slide28

☺ Mismatch repair

CH3

CH3

CH3

CH3

CH3

CH3

MutS/MutL

MutH

CH3

CH3

DNA Polymerase

DNA Ligase

hereditary nonpolyposis colorectal cancer hnpcc
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 breaks1
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)
hrr 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

HRR Vs. NHRR