The document discusses the mechanisms of DNA repair essential for maintaining genetic stability in cells, which include direct repair, excision repair, mismatch repair, recombination repair, and error-prone repair. Each mechanism involves specific processes and enzymes to address different types of DNA damage, with particular emphasis on repair pathways such as base excision repair and nucleotide excision repair. The document also highlights the implications of repair system failures, such as hereditary diseases linked to mutations in repair genes.

1. DNA Repair Mechanisms

2. Introduction
 The survival of the individual demands genetic
stability.
 Maintaining genetic stability requires
mechanisms for repairing the many accidental
lesions that occur continually in DNA.
 Without repair systems, a genome would not be
able to maintain its essential cellular functions.

3. Repair mechanisms:
1. Direct Repair (Reversal of damage)
2. Excision repair
3. Mismatch repair
4. Recombination repair
5. Error-prone repair
6. Restriction-modification systems

4. 1. Direct Repair
• Enzymatically un-do the damage
• a) Photoreactivation – light-
dependent direct system
• b) Removal of methyl groups -

5. 1a. Photoreactivation
• DNA Photolyase: when stimulated by light
with a wavelength b/w 300 & 500 nm, the
enzyme binds to pyrimidine dimers and
catalyzes a photochemical reaction
• Breaks the cyclobutane ring and reforms
two adjacent T’s
• 2 subunits, encoded by phrA and phrB.

6. Photolyase breaks apart pyrimidine dimers
N
HN
H
O
O
CH3
Thymine
N
HN
H
O
O
CH3
Thymine
d-ribose d-ribose
5
5
6 6 DNA
Photolyase
breaks the
bonds
between the
dTs

8. 2. Excision repair
• Excision repair involves excision of a segment
of the polynucleotide containing a damaged
site, followed by resynthesis of the correct
nucleotide sequence by a DNA polymerase.
• These pathway fall into two categories:
(a) Base excision repair
(b) Nucleotide excision repair

9. 2a. Base excision repair
• Base excision repair involves removal of a damaged
nucleotide base, excision of a short piece of the
polynucleotide & resynthesis with a DNA Polymerase.
• It is used to repair many minor change like alkylation,
deamination resulting from mutagen exposure.
• DNA glycosylase initiates the repair process; cleaves N-
glycosidic bonds, liberating the altered base & generating
an apurinic/apyrimidinic site (AP sites).
• AP sites repaired by an AP endonuclease repair pathway.
AP endonucleases introduce chain breaks by cleaving the
phosphodiester bonds at AP sites.
• This bond cleavage initiates an excision-repair process
further mediated by 3 further enzymes – an exonuclease,
DNA polymerase I & DNA ligase.

11. 2b. Nucleotide Excision Repair
• This repair system includes breaking of phosphodiester
bond on either side of the lesion, on the same strand,
resulting in the excision of an oligonucleotide.
• This excision leaves a gap that is filled by repair synthesis,
and a ligase seals the breaks.
• The uvr system of excision repair in E. coli is the best-
studied example.
• Enzymes involved in uvr system of repair: ABC
excinulease (3 subunits, products of uvrA, uvrB & uvrC
genes); UvrD (helicase II); DNA Polymerase I & DNA
ligase.

12. UvrABC excision repair
5'
3'
damaged site
5'
3'
A A
B
5'
3'
A A
B
+
(UvrA) UvrB recognizes the damaged site
2
(UvrA) dissociates
2
ATP

13. Cleavage and helicase
5'
3'
B
C
5'
3'
B
C
5'
3'
B
C
5'
3'
B
+
UvrC binds UvrB at the damaged site
UvrBC nicks both 5' and 3' to the damaged site
UvrD (helicase II) unwinds and liberates the damaged fragment
ATP
ATP

14. Fill in with polymerase and ligate
5'
3'
5'
3'
5'
3'
DNA polymerase I fills in the gap
dNTPs
NAD DNA ligase

15. Mutations in excision repair in eukaryotes can cause
Xeroderma Pigmentosum (XP), rare inherited
disease, inability of skin cells to repair UV-induced
DNA lesions (thymine dimer). Individuals suffering
from this autosomal recessive condition are
extremely sensitive to sunlight.
Human Analogous
Gene Protein Function to E. coli:
XPA Binds damaged DNA UvrA/UvrB
XPB Helicase, Component of TFIIH UvrD
XPC DNA damage sensor
XPD Helicase, Component of TFIIH UvrD
XPE Binds damaged DNA UvrA/UvrB
XPF Works with ERRCI to cut DNA UvrB/UvrC
XPG Cuts DNA UvrB/UvrC

16. 3. Mismatch repair
• Action of DNA polymerase III (including
proofreading exonuclease) results in 1
misincorporation per 108
bases synthesized.
• Mismatch repair reduces this rate to 1 change
in every 1010
or 1011
bases.
• Recognize mispaired bases in DNA, e.g. G-T
or A-C base pairs
• These do not cause large distortions in the
helix: the mismatch repair system apparently
reads the sequence of bases in the DNA.

17. Role of methylation in discriminating parental
and progeny strands
• DNA adenine methylase (Dam) converts ‘A’ to 6-
methyladenines in the seq. 5-GATC-3, and DNA
cytosine methylase (Dcm) converts ‘C’ to 5-
methylcytosines in 5-CCAGG-3 & 5-CCTGG-3.
• Methylation is delayed for several minutes after
replication.
• Mismatch repair works on the un-methylated
strand (which is newly replicated, the daughter
strand) so that replication errors are removed
preferentially.

18. Enzymes involved in Mismatch Repair
• E.coli involves 3 Mut proteins (MutH, MutL, MutS), coded
by the mut genes.
• MutS: recognizes the mismatch (heteroduplex)
• MutL: a dimer; in presence of ATP, binds to MutS-
heteroduplex complex to activate MutH
• MutH: endonuclease that cleaves 5' to the G in an
unmethylated GATC, leaves a nick.
• DNA helicase II & exonuclease I: combined action of both
removes a segment of the new strand b/w the cleavage
site & a point just beyond the mismatch.
• DNA Pol III fills the resulting gap.
• DNA ligase seals the nick.

19. MutH, L,
S action
in
mismatch
repair
#1

20. MutH, L, S action in mismatch repair #2

21. Mismatch repair: Excision of the
misincorporated nucleotide

22. Eukaryotic homologs in mismatch repair
• Human homologs to mutL (hMLH1) and
mutS (hMSH2, hMSH1) have been
discovered.
• Mutations in them can cause one of the
most common hereditary cancers,
hereditary nonpolyposis colon cancer
(HNPCC).

23. 4. Recombination repair:
• Recombination-repair is a post-replication
repair, process of filling a gap in one strand
of dsDNA by retreiving a homologous single
strand from another dsDNA.

24. 4. Recombination repair: retrieval of
information from a homologous single strand
5'
3'
damaged site
Recombination repair, a system for retrieval of information
Replication past a damaged site leaves a
gap on the opposite strand plus one
correct copy.
5'
3'
5'
3'
5'
3'
5'
3'
Gap is repaired by retrieving DNA from the correct copy of the chromosome,
using DNA recombination.
Gap in the correct copy is filled in by DNA polymerase.
Damaged site can be
repaired by excision repair
(e.g. UvrABC)

25. Repair of double strand DNA break
• Ionizing radiation, oxidizing agents, and
replication errors may cause double strand
break in DNA.
• 2 distinct mechanisms have evolved to
repair these damages –
(a) Non-homologous end joining
(b) Homologous end joining

26. Non-homologous end joining
• An emergency solution to the repair of double
strand break.
• In this process, broken ends are juxtaposed and
rejoined by DNA ligation, generally with the loss of
one or more nucleotides at the site of joining.
• Ku70 & Ku80 heterodimer recognizes the broken
ends. Ku proteins allow DNA dependent protein
kinase (DNA-PKcs) & Artemis to act.
• Artemis shows both endo- and exonuclease
activities & trims the DNA ends.
• DNA ligase IV in association with XRCC4, finally
joins the double strand ends.

27. Non-homologous end joining

28. 5. Error-prone repair
• Many cells have mechanisms that enable them to
synthesize DNA repair enzymes as an emergency response
to severe DNA damage.
• SOS reponse ("Save Our Ship") in E. coli is the best-
studied example.
• SOS response genes are induced when the bacterial chr. is
extensively damaged; many are involved in DNA repair &
mutagenesis.
• In E. coli, any block to DNA replication caused by DNA
damage produces a signal that activates RecA protein.
Activation of RecA causes autocatalytic proteolytic cleavage
of the LexA repressor. The LexA repressor regulates the
transcription of all the SOS genes.

29. SOS response is controlled by LexA and RecA
recA lexA target gene
LexA
RecA
LexA
LexA
e.g. recA, lexA, uvrA, uvrB, umuC
Repressed
SOS response is controlled by LexA and RecA
OFF
ON
recA lexA target gene
LexA
e.g. RecA, UvrA, UvrB, UmuC
de-repressed
RecA is activated in the presence of damaged DNA. It serves as a co-protease to activate a latent,
self-cleaving proteolytic activity in LexA, thereby removing the repressor from SOS inducible genes.
RecA
RecA RecA
RecA
+ cleaved LexA
active proteins

30. LexA, RecA in the SOS response

31. Error-prone repair system
• When the genome is subjected to heavy damage through
exposure to UV light or a DNA-damaging reagent, DNA
replication is halted because normal DNA replication with
DNA Polymerase III can’t proceed past many types of DNA
lesions.
• RecA also triggers cleavage of UmuD (UV mutagenesis)
protein.
• The cleavage event activates UmuD and the error-prone
repair system.
• UmuD2-UmuC complex binds to a RecA filament near the
site of damage, RecA activates the complex by cleaving
UmuD to generate UmuD*, and the complex (UmuD*2C or
DNA Polymerase V) then synthesizes a stretch of DNA to
replace the damaged material.

32. UmuC, UmuD in error-prone repair

33. Error-prone repair system
• Because proper base pairing is often impossible at
the site of a lesion, this translesion replication is
error-prone.
• DNA Pol. V, a Y-family DNA Pol., lack 3 5
proofreading exonuclease activity is also known as
error-prone DNA polymerase.