DNA REPAIR MECHANISM

1. MECHANISM OF DNA
REPAIR
Dr Sony Nanda
MCh Resident ,Dept of Gynec Oncology
AHPGIC Cuttack.

4. genetics.gsk.com/ graphics/dna-big.gif
DNA Structure
• Four nucleotides: adenine (A), cytosine
(C), guanine (G), and thymine (T)
• Bindings:
• A with T (weaker), C with G (stronger)
• Forms a double helix – each strand is
linked via sugar-phosphate bonds (strong),
strands are linked via hydrogen bonds
(weak)
• Genome is the part of DNA that encodes
proteins:
• …AACTCGCATCGAACTCTAAGTC…

5. DNA Sequence Analysis
5

6. Nucleotide=base+sugar+phosphate
COMPOSITION DNA
PURINE BASES Adenine Guanine
PYRIMIDINE
BASES
CytosineThymine
PENTOSE SUGAR 2-deoxyribose
INORGANICACID PhosphoricAcid

7. DNA DAMAGE & MECHANISMS OF REPAIR

8. DNA DAMAGE
• DNA damage affects the primary structure of the double helix; that is, the bases
themselves are chemically modified.
• These modifications can in turn disrupt the molecules' regular helical structure by
introducing non-native chemical bonds or bulky adducts that do not fit in the
standard double helix.
• DNA is, however, supercoiled and wound around "packaging" proteins
called histones (in eukaryotes), and these superstructures are vulnerable to the
effects of DNA damage

9. SOURCES:
• DNA damage can be subdivided into two main types:
1.endogenous damage such as attack by reactive oxygen species produced from normal metabolic
byproducts (spontaneous mutation)
2.exogenous damage caused by external agents
1. ultraviolet [UV 200–400 nm] radiation from the sun or other artificial light sources
2. x-rays and gamma rays
3. thermal disruption
4. human-made mutagenic chemicals, especially aromatic compounds that act as DNA intercalating agents
• The replication of damaged DNA before cell division can lead to the incorporation of wrong bases
opposite damaged ones.
• Daughter cells that inherit these wrong bases carry mutations from which the original DNA sequence
is unrecoverable .

11. TYPES OF DNA REPAIR MECHANISM
• DIRECT REVERSAL
• SINGLE STRAND BREAK
• DOUBLE STRAND BREAK
• TRANSLESION SYNTHESIS
GENETIC ALTERATIONS IN TWO OF THESE REPAIR MECHANISMS ARE
ESSENTIAL IN GYNECOLOGIC MALIGNANCIES.
- SS REPAIR(MMR )
-1 DS REPAIR(BRCA 1-2)

12. [A] DIRECT REVERSAL
• Cells are known to eliminate three types of damage to their DNA by chemically
reversing it.
• These mechanisms do not require a template, since the types of damage they
counteract can occur in only one of the four bases.
• Such direct reversal mechanisms are specific to the type of damage incurred and do
not involve breakage of the phosphodiester backbone.
• Mostly seen in prokaryotes

13. Types of Direct Damage & Reversal
1.Formation of pyrimidine dimers upon irradiation with UV light (Abnormal covalent
bond between adjacent pyrimidine bases – cytosine & thiamine. )
The photoreactivation process directly reverses this damage by the action of the
enzyme photolyase.
[Photolyase, an old enzyme present in bacteria, fungi, and most animals no longer functions in humans, who
instead use nucleotide excision repair to repair damage from UV irradiation]
2.Another type of damage, methylation of guanine bases, is directly reversed by the
protein methyl guanine methyl transferase (MGMT).
[This is an expensive process because each MGMT molecule can be used only once.]
3. Another type of DNA damage reversed by cells is certain methylation of the bases
cytosine and adenine.

14. [B] SINGLE CHAIN BREAKS REPAIR
When only one of the two strands of a
double helix has a defect, the other strand
can be used as a template to guide the
correction of the damaged strand.
TYPES are:
1.BASE EXCISION REPAIR
2.NUCLEOTIDE EXCISION REPAIR
3.MISMATCH REPAIR

15. B1.BASE EXCISION REPAIR

16. BASE EXCISION REPAIR
Damaged single bases or nucleotides are most commonly
repaired by removing the base or the nucleotide involved
and then inserting the correct base or nucleotide.
• In base excision repair, a glycosylase enzyme removes the
damaged base from the DNA by cleaving the bond
between the base and the deoxyribose.
• These enzymes remove a single base to create an apurinic
or apyrimidinic site (AP site).
• Enzymes called AP endonucleases nick the damaged
DNA backbone at the AP site.
• DNA polymerase then removes the damaged region using
its 5’ to 3’ exonuclease activity and correctly synthesizes
the new strand using the complementary strand as a
template.
• The gap is then sealed by enzyme DNA ligase

17. B2. NUCLEOTIDE EXCISION REPAIR

18. NUCLEOTIDE EXCISION REPAIR
• Bulky, helix-distorting damage, such
as pyrimidine dimerization caused by
UV light
• usually repaired by a 3-step process.
1.damage is recognized
2. 12-24 nucleotide-long strands
of DNA are removed both upstream
and downstream of the damage site
by endonucleases
3.the removed DNA region is then
resynthesized.

19. B3.MMR

20. MISMATCH REPAIR
• Highly conserved mechanism
• Recognises & repairs erroneous nucleotide insertions ,deletions , misincorporations.
• Analysed in E.coli
-Inactivation results in hypermutable E.,coli strains
-Due to 50-100 fold increased mutations these proteins named Mut (Mut S ,Mut H,Mut L)
• Human cells
-3 Mut S Homologs (MSH2,MSH3,MSH6)
-3 Mut L Homologs(MLH1,PMS1,PMS2)
-No eukaryotic homolog for Mut H.

21. • Eukaryotic MMR proteins function as heterodimer & show slightly different repair
mechanism.
• MSH2/MSH6 Heterodimer repairs
-base substitution
-Smaller DNA loops
• MSH2/MSH3 heterodimer repair large DNA loops.

22. DNA SS- MMR
(Steps)
MSH2/MSH6 or MSH2/MSH3 heterodimer recognise
DNA base pair mismatch
1.Form a ring around mismatch recognition domains.
2.Recruit MSH/MLH heterodimer
which assemble larger protein machinery for repair of
mismatch.
3.DNA repair
-strand discrimination by PCNA(Proliferating Nuclear
Cell Antigen)
-unwinding of DNA by DNA helicase
-excision of mismatch containing DNA portion by
EXO1(Exonuclease 1)
4 .Synthesis by DNA polymerases (POL)

23. MSI :
Alteration in mismatch repair proteins result in
increased mutation rate :
-genetic instability (general)
-instability of microsatellites(short nucleotide
repeats .)(in particular).
Microsatellites are short repeats in DNA
sequence (esp. dinucleotide repeats of Adenine &
Cytosine)
-Longer tandem repeats called mini satellites
-Even longer Satellites
Microsatellites located in introns(intragenic
regions )-non translated DNA regions .

24. • Microsatellites vary from person to person &
contributes to individual Dna Fingerprint
• MSI used in Colon & Endometrial cancer.
• MSI caused by
-germline mutations in MMR protein.
-epigenetic silencing by hypermethylation.
-downregulation of MMR Mrna by microRNAs.

25. [C] DOUBLE-STRAND BREAKS
• Double-strand breaks, in which both strands in the double helix are severed, are
particularly hazardous to the cell because they can lead to genome rearrangements.
• 3 mechanisms exist to repair double-strand breaks (DSBs):
non-homologous end joining (NHEJ),
microhomology-mediated end joining (MMEJ),
homologous recombination (HR)

27. C1.HOMOLOGOUS RECOMBINATION
600 × 708

28. HOMOLOGOUS RECOMBINATION [HR]
• Homologous recombination requires the presence of an identical or nearly identical
sequence to be used as a template for repair of the break.
• This pathway allows a damaged chromosome to be repaired using a
sister chromatid (available in G2 after DNA replication) or a homologous
chromosome as a template.

29. Steps in HR Pathway
1. DSB recognition(ATM/ATR kinases)
• These kinases phosphorylate & activate CHEK 2,
TP53,BRCA1.
• BRCA1 will serve as a scaffold to organise repair
protein including BARD1 & BRIP1.
2. MRN complex resects damaged DNA site in 5’ to
3’ direction in order to produce over hanging ends of
ss DNA
3. Sister chromatid searched for homologous DNA.
4. RAD 51 invades homologous DNA strand &
forms a displacement loop.(D loop)
5. Following D loop formation DNA synthesized
using sister chromatid DNA as a template.
6. Holliday junctions resolved mostly .
7. Newly synthesized DNA is ligated.

30. • ATM-ATAXIA TELANGIECTASIA MUTATED
• ATR-ATAXIA TELANGIECTASIA & RAD3 RELATED PROTEIN
• CHEK2 –CHEKPOINTB2
• BARD1-BRCA ASSOCIATED RING DOMAIN PROTEIN 1
• BRIP1-BRCA INTERACTING PROTEIN 1
• MRN COMPLEX-MRE11+RAD50+NBN
• PALB2-PARTNER &LOCALISER OF BRCA 2.

31. C2.NON HOMOLOGOUS END JOINING

32. NON HOMOLOGOUS END JOINING(NHEJ)
• In NHEJ, DNA Ligase IV, a specialized DNA
ligase that forms a complex with the
cofactor XRCC4, directly joins the two ends.
• To guide accurate repair, NHEJ relies on short
homologous sequences called microhomologies
present on the single-stranded tails of the DNA
ends to be joined.
• If these overhangs are compatible, repair is
usually accurate.
• NHEJ can also introduce mutations during
repair.
• NHEJ is especially important before the cell has
replicated its DNA, since there is no template
available for repair by homologous
recombination.

34. C3.MICROHOMOLOGY MEDIATED END
JOINING

35. MICROHOMOLOGY MEDIATED END
JOINING
• MMEJ starts with short-range end
resection by MRE11 nuclease on either side of a
double-strand break to reveal microhomology
regions.
• In further steps,Poly (ADP-ribose) polymerase
1 (PARP1) is required and may be an early step in
MMEJ.
• There is pairing of microhomology regions
followed by recruitment of flap structure-specific
endonuclease 1 (FEN1) to remove overhanging
flaps.
• This is followed by recruitment of XRCC1–
LIG3 to the site for ligating the DNA ends, leading
to an intact DNA.
• MMEJ is always accompanied by a deletion, so
that MMEJ is a mutagenic pathway for DNA repair

37. DNA DAMAGETOLERANCE

39. [D1]TRANSLESION SYNTHESIS [TLS]

40. • When cells cannot repair themselves a failsafe mechanism that allows the replication
machinery to bypass the dna damaged site called translesion synthesis occurs
• Catalysed by special class of DNA polymerases that synthesize DNA directly across the
damaged site
• Translesion polymerase is produced by cell in response to the DNA damage.

41. [D2]TEMPLATE SYNTHESIS [TS]
• The TLS mechanism has been characterized as error-prone due to the deficient
proofreading activity of the TLS polymerase, which increases the risk of mutation.
• Not surprisingly, TLS is a major source of cellular mutagenesis.
• In contrast, another mode of DDT, the template switching (TS), involves
recombination to a homologous DNA template on a sister chromatid, which is
similar to the HR process and is believed to be more accurate in the outcome than
TLS .
• The repair activities of TLS and TS start behind the replication fork, suggesting that
they could occur during or after DNA replication, with TS beginning earlier in the S
cell-cycle phase and TLS in the late S phase.

42. USES IN GYNECOLOGIC ONCOLOGY

43. USES INVARIOUS CANCERS

44. Inhibitors of DNA repair-
PARP inhibitors.
• After DNA damage,enzymes PARP 1& 2 bind to
the damaged DNA & recruit DNA repair proteins .
• Continued Autoparylation results in destablisation
of the PARP/DNA complex & eventually the
dissociation of PARP from the DNA.
• This allows NER ,BER ,DNArepair proteins to
bind to DNA damage site.
• Using PARP inhibitors PARP enzymes remain
bound to site of DNA damage & are trapped.(PARP
Trapping)
• Because of PARP trapping DNA repair proteins
cant bind.
• Hence unpaired SS breaks may degenerate into DS
breaks which cant be repaired in absence of an
intact HR repair pathway.

45. • PARP inhibitors have also demonstrated certain limitations .
1.Predominantly, the varying PARP trapping ability by different PARP inhibitors
potentially lead to the off-target PARP trapping on the DNA of normal cells.
2.Besides, the emerging resistance to PARP inhibitors also poses challenges to their
clinical application,
Mechanisms of resistance:
 loss of PARP trapping,
 upregulated drug efflux protein expression,
 stabilized replication fork stabilization
 the restoration of HR pathway

46. WHATS NEW……………

47. Poly(ADP-ribose) glycohydrolase (PARG)
• The above limitations of PARP inhibitors
motivated the design of additional
therapeutic targets for PARPi-resistant
tumors.
• PARG reverses the action of PARP
enzymes by hydrolyzing the ribose–ribose
bonds in PAR following DNA
damage.(Structural change in PAR
enzymes)
• Likewise, the active role of PARG in
DNA replication and repair leads to
increased sensitivity to DNA damaging
agents in PARG-deficient cells.

50. THANKYOU