dna damage and reapair mechanisms easyly unterstood

1. DNA DAMAGE AND REPAIR
By
M.SyedMarjuk.,B.Sc.,hdca.,dhn
DNA Damage And Repair

2. • DNAin the living cell is subjected tomany
chemical alterations.
• Thegenetic information encoded in theDNA
hasto remainuncorrupted
• Any chemical changesmust be corrected.
• A failure to repair DNAproducesamutation.
DNA Damage And Repair

3. • Radiations
oHighlyreactive oxygenradicalsproduced during
normal cellular respiration aswell asby other
biochemical pathways
oIonizingradiation suchasgammarays and x-rays
oUltraviolet rays,especially the UV-Crays (~260
nm) that are absorbed strongly by DNAbut alsothe
longer-wavelength UV-Bthat penetrates the ozone
shield
DNA Damage And Repair

4. • Chemicalsin the environment
oAromatic hydrocarbons, including somefound
in cigarette smoke
oPlant and microbial products, e.g.the
Aflatoxin produced in moldypeanuts
oChemicals used in chemotherapy, especially
chemotherapy of cancers.
DNA Damage And Repair

5. DNArepair canbe grouped into two major
functional categories:
A)Direct Damagereversal
B)Excisionof DNAdamage
DNA Damage And Repair

6. Thedirect reversal of DNAdamage is by far the
simplest repair mechanism that involves asingle
polypeptide chain, with enzymatic propertieswhich
binds to the damage and restores the DNAgenome
to its normal state in asingle-reaction step. The
major polypeptides involved in this pathwayare:
i) DNAphotolyases, the enzymesresponsible for
removing cyclobutane pyrimidine dimers from DNA
in alight-dependent process called asphoto
reactivation
DNA Damage And Repair

7. •
ii) O6-methylguanine-DNA
methyltransferase I andII
(MGMT), alsocalledDNA-
alkyltransferases,remove
the modified baseslikeO6-
alkylguanine and O4-
alkylthymine.
Thephotolyase protein is
notfound in all living cells.
However, the DNA-
alkyltransferases are
widespread in nature.
DNA Damage And Repair

8. i) Baseexcision repair (BER)
ii) Nucleotide excision repair (NER),
iii) Mismatch repair (MMR)and
iv) Strand break repairs.
•
• In these reactions anucleotide segment containing
basedamage, double-helix distortion or mispaired
basesis replaced by the normal nucleotide sequencein
anew DNApolymerase synthesis process.
All of these pathways have been characterized inboth
bacterial and eukaryotic organisms.
DNA Damage And Repair

9. • BERis initiated by DNAglycosylases,which
catalyze the hydrolysis of the N-glycosidic
bonds, linking particular types of chemically
altered basesto thedeoxyribose-phosphate
backbone.
• DNAdamage is excisedasfree bases,
generating sites of baseloss called apurinicor
apyrimidinic (AP)sites.
DNA Damage And Repair

10. •
•
•
•
TheAPsites are substrates for APendonucleases.
Theseenzymes produce incisions in duplex DNAasa
result of the hydrolysis of aphosphodiester bond
immediately 5' or 3' to eachAPsite.
Theribose-phosphate backbone is then removed from
the DNAthrough the action of aspecific exonuclease
called deoxy ribophosphodiesterase or dRpase.
Finally, the DNApolymerase and aligase catalyze the
incorporation of aspecific deoxyribonucleotide into
the repaired site, enabling correct basepairing
DNA Damage And Repair

11. Baseexcision-repairofDNA
•
•
•
•
• Theenzyme uracil DNA
glycosylase removes the
uracil created by
spontaneous deaminationof
cytosine in the DNA.
An endonuclease cutsthe
backbone near the defect
An endonuclease removes a
few bases
The defect is filled in by the
action of a DNApolymerase
and
Thestrand is rejoined bya
ligase.
DNA Damage And Repair

12. •
•
•
•
Thismechanism is used to replace regions of damagedDNA
up to 30 basesinlength.
Common causesof such DNAdamage include ultraviolet
(UV) light, which induces the formation of cyclobutane
pyrimidine-pyrimidine dimers, and smoking, whichcauses
formation of benzo[a]pyrene-guanineadducts.
Ionizing radiation, cancer chemotherapeutic agents, anda
variety of chemicals found in the environment cause base
modification, strand breaks, cross-linkage between bases
on opposite strands or between DNAand protein, and
numerous other defects.
Theseare repaired by aprocess called nucleotide excision-
repair
DNA Damage And Repair

13. • NERis a much more complex biochemical
process than BER,especially in eukaryotic
cells.
• Several gene products are required in a
multiple step process, during which the
ordered assembly of DNAproteins providesan
enzymatic complex that discriminates
damagedfrom undamagedDNA.
DNA Damage And Repair

14. (NER)
•
•
•
In eukaryotic cells theenzymes
cut between the third to fifth
phosphodiester bond 3' from
the lesion, and on the 5' side
the cut is somewhere between
the twenty-first and twenty-
fifth bonds.
Thus,afragment of DNA27–29
nucleotides long isexcised.
After the strand is removed itis
replaced, again by exact base
pairing, through the action of
yet another polymeras e,and
the endsare joined to the
existing strands by DNAligase.
DNA Damage And Repair

15. •
• In Escherichia
coli there are three
specific proteins,
called UvrA, Band C,
involved in lesion
recognition and
endonuclease incision.
This fragment is
released by UvrD
helicase action,
generating agapthat
is finally submittedto
repair synthesis
DNA Damage And Repair

16. • Nucleotide-excision repair proceeds most
rapidly in cells whose genesare beingactively
transcribed on the DNAstrand that is serving
asthe template for transcription.
• If RNApolymerase II, tracking along the
template (antisense) strand), encounters a
damagedbase, it can recruit other proteins,to
make aquick fix before it moves on to
complete transcription of thegene.
DNA Damage And Repair

17. • Mismatch repair corrects errors made whenDNA
is copied.
• For example, a Ccould be inserted opposite an A,
or the polymerase could slip or stutter and insert
two tofive extra unpaired bases.
• Specific proteins scanthe newly synthesized
DNA,using adenine methylation within aGATC
sequenceasthe point of reference
• Thetemplate strand is methylated, andthe
newly synthesized strand isnot.
DNA Damage And Repair

18. • Thisdifference allows the repair enzymesto
identify the strand that contains the errant
nucleotide which requiresreplacement.
• If amismatch or small loop is found, aGATC
endonuclease cuts the strand bearing the
mutation at asite corresponding to theGATC.
• An exonuclease then digests this strand from the
GATCthrough the mutation, thus removing the
faulty DNA.Thiscanoccur from either end if the
defect is bracketed by two GATCsites.
• Thisdefect is then filled in by normalcellular
enzymesaccording to basepairing rules
DNA Damage And Repair

19. •
•
• This mechanism corrects a
single mismatch base pair
(eg, Cto Arather than Tto
A)or ashort regionof
unpaired DNA.
Thedefective region is
recognized by an
endonuclease that makesa
single-strand cut at an
adjacent methylated GATC
sequence.
TheDNAstrand is removed
through the mutation,
replaced, and religated.
DNA Damage And Repair

20. •
•
•
• In E coli, three proteins (Mutt S,
Mutt L,and Mutt H)are required
for recognition of the mutation and
nicking of thestrand.
Other cellular enzymes,including
ligase, polymerase, and SSBs,
remove and replace the strand.
Theprocessis more complicated in
mammalian cells, asabout six
proteins are involved in the first
steps.
Faulty mismatch repair hasbeen
linked to hereditarynonpolyposis
colon cancer (HNPCC),one of the
most common inherited cancers.
DNA Damage And Repair

21. • Ionizing radiation and certain chemicals can
produce both single-strand breaks (SSBs)and
double-strand breaks (DSBs)in the DNA
backbone.
i) Single-StrandBreaks(SSBs)
• Breaks in a single strand of the DNAmolecule
are repaired using the same enzyme systems
that are used in Base-ExcisionRepair(BER).
DNA Damage And Repair

22. ii) Double-StrandBreakRepair
• There are two mechanisms by which the cell
attempts to repair acomplete break inaDNA
molecule:
• 1) Direct joining of the broken ends.Thisrequires
proteins that recognize and bind to the exposed
ends and bring them together for ligating. This
type of joining is also calledNonhomologous
End-Joining(NHEJ).Aprotein called Ku is
essential for NHEJ.
DNA Damage And Repair

23. • Errors in direct joining may be acauseofthe
various translocationsthat are associated
with cancers.Examples:
• Burkitt's lymphoma
• Philadelphia chromosome in chronic
myelogenous leukemia (CML)
• B-cell leukemia
DNA Damage And Repair

24. 2) HomologousRecombination.Here the broken ends
are repaired using the information on theintact
•
•
•
•
•
sisterchromatid,or on the
homologous chromosome
samechromosomeif there are duplicate copies of the
gene on the chromosome oriented in opposite
directions (head-to-head or back-to-back).
Two of the proteins used inhomologous
recombination are encoded by the
genesBRCA1 and BRCA2.
Inherited mutations in these genespredisposewomen
to breast and ovariancancers.
DNA Damage And Repair

25. Meiosisalsoinvolves
DSBs
Recombination
between homologous
chromosomes in
meiosis I also involves
the formation ofDSBs
and their repair.
Meiosis I with the
alignment of
homologous
sequencesprovidesa
mechanism for
repairing damaged
DNA.
DNA Damage And Repair

26. Thank you
DNA DAMAGE AND REPAIR