1. Presented by - Amit Tiwari

2. What is DNA?
• A polymeric molecule consisting of
deoxyribonucleotide building blocks that in a
double-stranded, double-helical form is the
genetic material of all living organisms.
• Each nucleotide consists of a pentose (five-
carbon) sugar, a nitrogenous (nitrogen-
containing) base (usually just called a base), and
a phosphate group.

4. STRUCTURE OF DNA

5. How DNA is damaged?
DNA can be damaged in number of ways:
• Exposure to UV light
• Mechanical shearing
• Phenol extraction
• Dessication
• Heating

7. DNA Repair
• The process by which a cell uses a series of
special enzymes to repair mutations (changes)
in DNA and restore the DNA to its original
state.
• Maintaining the integrity of the information in
DNA is a cellular imperative, supported by an
elaborate set of DNA repair systems.

8. Types of DNA Repair
• SOS Repair ( Error – prone repair system )
• Base Excision Repair
• Nuclotide Excision Repair
• Repair of Alkylation Damage

9. SOS RESPONSE
• A system that repairs severely damaged bases
in DNA by base excision and replacement,
even if there is no template to guide base
selection.
• This process is a last resort for repair and is
often the cause of mutations.

10. Historical Overview of SOS Response
• Jean Weigle in 1953 observed reactivation of UV –
irradiated lambda phage increased when irradiated
phages were plated on previously irradiated
Escherichia coli cells ( Weigle reactivation )
• Miroslav Radman concluded that in Escherichia coli
there is DNA repair system dependent on LexA and
RecA proteins and named it as “ SOS repair ’’

11. Recombination Proteins
• recA and lexA genes were first to be
recognized as being involved in SOS induction
• 27 kDa LexA and 36 kDa RecA proteins were
known as Recombination proteins operates in
sexual life and genetic exchange of bacteria
• RecA protein participates in genetic DNA
exchange , in recF , recO , recR , recN and
ruvABC- dependent recombinational DNA
repair

12. Structure of LexA and RecA

13. SOS INDUCED DNA POLYMERASES
• Five different DNA polymerases found in Escherichia
coli : pol 1 to pol 5
• Pol I - fills gaps in course of DNA repair and in
discontinuous DNA synthesis on lagging strand
• Pol III - DNA polymerizing enzyme
• Pol II - reactivates replicative DNA complex
• Pol IV - induces of mutations in lambda phage and in
episom F’
• Pol V - error prone translesion DNA polymerase

15. Mechanism of SOS Induction
• In presence of ssDNA RecA protein interacts
with LexA protein
• LexA protein represses about 18 genes
including itself
• Each of those 18 genes has consensus
sequence in their promoter called SOS box :-
5’-CTGX10CAG-3’
where X10 refers to any 10 bases

17. Mechanism of SOS Induction
• LexA protein binds to SOS box and limits the
trancription of genes
• When RecA is activated it interacts with LexA
to trigger autocatalytic properties of LexA

18. Mechanism of SOS Induction
• RecA is not activated as there is no ssDNA ,
resulting in no longer destruction of LexA
protein
• LexA represses the suite of proteins involved
in SOS response and SOS response is over.

20. Base Excision Repair
• An enzyme-catalyzed process for repairing
damaged DNA by removal of the altered base,
followed by excision of the baseless
nucleotide.
• The correct nucleotide then is inserted in the
gap.

21. Base Excision Repair
• Major pathway for repair of modified bases ,
uracil misincorporation , oxidative damage
• Base can be removed from nucleotide within
DNA : by direct action of agents such as
radiation , by spontaneous hydrolysis , by an
attack of oxygen free radicals , or by DNA
glycosylases.

22. DNA glycosylases
• A family of DNA repair enzymes that recognize
damaged nucleotide bases and remove them
by hydrolyzing the N-glycosidic bond that
attaches them to the sugar backbone of
the DNA molecule.

23. Types of DNA glycosylases
• Uracil DNA glycosylase
• Helix - hairpin - helix glycosylase
• 3-methyl-purine glycosylase (MPG)
• Endonuclease VIII-like glycosylase

24. Uracil DNA glycosylase
E. coli Uracil-DNA
Glycosylase (UDG)
catalyses the release of
free uracil from uracil-
containing DNA. UDG
efficiently hydrolyzes
uracil from single-
stranded or double-
stranded DNA, but not
from oligomers
(6 or fewer bases).

25. Helix – Hairpin – Helix glycosylase
Structure of selected HhH Superfamily members
HhH motif was first discovered in Endo III as a sequence –
independent DNA binding motif.

26. 3-methyl-purine glycosylase (MPG)
• Methylpurine-DNA glycosylase (MPG, or
alkyladenine DNA glycosylase (AAG)) is a base
excision-repair protein, catalyzing the first step in
base excision repair by cleaving damaged DNA
bases within double-stranded DNA to produce an
abasic site.
• MPG bends DNA by intercalating between the
base pairs, causing the damaged base to flip out
of the double helix and into the enzyme active
site for cleavage.

27. 3-methyl-purine glycosylase (MPG)
• It is responsible for the hydrolysis of the
deoxyribose N-glycosidic bond, excising 3-
methyladenine and 3-methylguanine from
damaged DNA.

28. Endonuclease VIII-like glycosylase
• Recognizes and repairs oxidized pyrimidines in
the Base Excision Repair (BER) pathway.

29. AP endonuclease
• Recognize and promote repair of AP sites
generated either spontaneously or as the first
step in BER.
• These process the products of both
monofunctional DNA glycosylases, which
produce abasic sites, and the glycosylase/ AP
lyases which cleave both base – to generate
an abasic site.

30. AP endonuclease
• It also cleaves the phosphodiester DNA
backbone – to produce a 5’ phosphate and a
3’ αβ - unsaturated aldehyde group.

31. Mechanism of BER

32. Mechanism of BER

33. Mechanism of BER

34. Nucleotide Excision Repair
• An enzyme-catalyzed process for removal of
thymine dimers from DNA and synthesis of a
new DNA segment complementary to the
undamaged strand.
• NER is important excision mechanism that
removes DNA damage induced by UV.

35. Formation of DNA adducts
• UV DNA damage results in bulky DNA adducts
• These adducts are thymine dimers and 6,4-
photoproducts

36. NER system proteins
NER involves four proteins:
• UvrA encoded by uvrA gene
• UvrB encoded by uvrB gene
• UvrC encoded by uvrC gene
• UvrD encoded by uvrD gene

37. UvrA and UvrB protein
structure

38. Mechanism of NER
• UvrAB scans and finds DNA damage
• UvrAs released; UvrC binds

39. Mechanism of NER
• Cuts made 5’ and 3’ to damage
• UvrD binds and unwinds region between cuts,
releasing the damaged segment.

40. Mechanism of NER
• DNA polymerase I fills in gap.
• DNA ligase joins the DNA segments; repair is
complete.

41. Repair of Alkylation Damage
• Alkylating agents transfer alkyl groups usually
methyl or ethyl group onto the bases at
various locations , such as oxygen of carbon-6
in guanine.
• In Escherichia coli , alkylation damage can be
repaired by an enzyme called O6-
Methylguanine-DNA methyltransferase,
encoded by the ada gene.

42. O6-Methylguanine-DNA
methyltransferase
• O6-Methylguanine-DNA methyltransferase
(MGMT) is a suicide enzyme that repairs the
pre-mutagenic, pre-carcinogenic and pre-toxic
DNA damage O6-methylguanine.
• It also repairs larger adducts on the O6-
position of guanine, such as O(6)-[4-oxo-4-(3-
pyridyl)butyl]guanine and O6-
chloroethylguanine.

43. O6-Methylguanine-DNA
methyltransferase

44. Repair of Alkylation Damage
• O6-Methylguanine-DNA methyltransferase
recognizes O6-Methylguanine in DNA and
removes methyl group, thereby changing the
base back to its original form.
• Mutations of the genes encoding these repair
enzymes result in a much higher rate of
spontaneous mutation.

45. THANK YOU!