For DNA

information must be transmitted

intact to daughter cells

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DNA Repair

Accuracy is maintained by:
1- High fidelity in replication
3- exonuclease activity of DNA pol I
Uracil-DNA N-glycosylase pathway
(corrects mutations from deamination of
cytosine) cytosine Uracil
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Accuracy is maintained by:
2-Mechanisms for correcting genetic info. in
damaged DNA

e.g due to chemical modifications
Irradiation changes

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Categories of DNA Repair

1. Mismatch Repair (Synthesis + Repairing)
MM created by replication errors
DNA Pol III proof reading
non-homologous recombination are recognized
and corrected

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DNA Pol III
Categories of DNA Repair
2. Base Excision Repair (Euk/Pro)
Starts at cleavage of glycosidic bond (connects
base to sugar-phosphate backbone)


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glycosidic bond
Categories of DNA Repair
3. Direct Repair - Damaged
base undergoes a chemical/UV
reaction Restores original
structure (pro)
e.g. DNA photolyase - E.coli

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Categories of DNA Repair
4. Nucleotide Excision Repair (Prok: 12/Euk: 28)
- damaged DNA:
excised
replaced with normal DNA

5. Recombinational Repair
- Fills gaps in DNA :
- Newly replicated DNA duplexes undergo genetic
recombination
Removal of damaged segment

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DIRECT DNA DAMAGE AND REPAIR

A variety of irradiation (ionizing, ultraviolet, etc)
DNA damage of a variety of sorts:
U.V. induced formation of Thymine Dimmer
Blocked replication and gene expression until
repaired
Prohotoreactivation enzyme
Photolyase
Prokaryote
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DNA REPAIR
(1) Photoreactivation (Light Repair)
UV induced formation of Thymine Dimer
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C T
Photoreactivation (Light Repair)
PHR/PRE gene
codes for photolyase
with cofactor folic acid
binds in dark to T dimer
When light shines on cell
folic acid absorbs the light (photon)
uses the energy to break bond of T dimer
photolyase then falls off DNA

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DNA REPAIR
(2) Excision Repair
(aka Dark Repair)
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Excision Repair (Dark Repair)

3 different types of repair mechanisms
use different enzymes

(a) AP Repair (Base Excision Repair, BER)
(b) UV Damage Repair (also called NER -
nucleotide excision repair)
(c) Mismatch Repair (MMR)

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(a) AP Repair
(Base Excision Repair, BER)

Repair of apurinic and apyrimidinic sites on DNA
in which base: has been removed
Base removed by:
DNA glycosylases
which remove damaged bases
ung gene codes for uracil-DNA glycosylase
recognizes and removes U in DNA
by cleaving the sugar-nitrogen bond to remove the base


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AP endonucleases:
class I nick at 3' side of AP site
class II nick at 5' side of AP site

Exonuclease removes short region of
DNA

DNA Pol I and ligase fill in gap

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(b) UV Damage Repair (also called NER -
nucleotide excision repair)
It uses different enzymes
NER removes a large "patch" around the
damage
Even though there may be only a single "bad"
base to correct, its nucleotide is removed along
with many other adjacent nucleotides
NER: UV
BER: Chemicals/Agents
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NER (UV Damage Repair)
Nuclease:
can detect T dimer
nicks DNA strand on 5' end of dimer
(composed of subunits coded by uvrA, uvrB
and uvrC genes)
UvrA protein and ATP bind to DNA at the
distortion
UvrB binds to the UvrA-DNA complex and
increases specificity of UvrA-ATP complex for
irradiated DNA
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UvrC nicks DNA 8 bases upstream and 4 or 5
bases downstream of dimer
UvrD (DNA helicase II; same as DnaB)
separates strands to release 12-bp segment
DNA polymerase I now fills in gap in 5'>3'
direction
ligase seals
polA - encodes DNA pol I
mutant was viable retained normal 5'>3'
exo activity
only 2% of polymerase activity

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Excision Repair of Thymine dimers by UvrABC
exinuclease of E.coli

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(c) Mismatch Repair (MMR)
Accounts for 99% of all repairs
Mismatch from replication
behind replication fork
Two ways to correct mistakes made during replication:
1) 3'>5' exonuclease - proofreading
2) Mismatch repair
mutL
mutS
mutH
and mutU (same UvrD) gene products involved (mut for
mutator because if gene is mutated, cell has increased
levels of spontaneous mutations)
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How does system recognize progeny strand
rather than parent strand as one with
mismatch?
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Because of methylation
DNA methylase (coded for by dam [DNA adenine methylase]
locus)
methylates 5'-GATC-3' sequence in DNA at A residue
Mismatch from replication recognized by mutL and mutS gene
products
mutH gene product nicks DNA strand (progeny strand) on
either side of mismatch
DNA helicase II from mutU gene (also called uvrD gene)
unwinds DNA duplex and releases nicked region
Gap filled in by DNA Pol I and ligase

DNA REPAIR
(1) Photoreactivation (aka Light Repair)

(2) Excision Repair (aka Dark Repair)

(3) Postreplicative (Recombinational)
Translation Bypass Repair



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DNA REPAIR
(3) Postreplicative
(Recombinational)
Translation Bypass
Repair

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SOS response
If T dimer is not repaired
DNA Pol III can't make complementary strand during
replication
leaves large gap (800 bases)
Gap may be repaired by enzymes in recombination system
RecA - coats ssDNA
it also acts as autocatalysis of LexA repressor
recA mutants - very UV-sensitive
Now have sister-strand exchange - a type of recombination
Translation bypass
Postreplicative repair is part of SOS response
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SOS Response

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LexA normally represses about 18 genes
sulA and sulB, activated by SOS system
inhibit cell division in order to increase amount of
time cell has to repair damage before replication
Each gene has SOS box in promoter
LexA binds SOS box to repress expression
RecA : LexA catalyses its own breakdown when RecA
is stimulated by ssDNA
due to RecA binding ssDNA in lesions
could then bind to DNA Pol III complex passing
through this area of the DNA and inhibit 3'>5'
exonuclease (proofreading) ability
RecA no longer catalyzes cleavage of LexA (which is
still being made)
so uncleaved LexA accumulates and turns the SOS
system off
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Why are DNA Repair Systems Necessary?

E.coli


Xeroderma Pigmentosum (XP)


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E.coli
repairing thymine dimers
important to bacteria
an E. coli strain that is:
phr (no photoreactivation)
recA (no translation by pass or SOS)
uvrA (no excision repair) is killed by a single
thymine dimer
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Xeroderma Pigmentosum (XP)
XP is a rare inherited disease of humans
predisposes the patient to:

pigmented lesions on areas of the skin exposed to
the sun
an elevated incidence of skin cancer


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Xeroderma Pigmentosum

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It turns out that XP can be caused by mutations in
any one of several genes
all of which have roles to play in NER
Some of them:

XPA, which encodes a protein that binds the
damaged site
assemble the other proteins needed for NER
XPB and XPD, which are part of TFIIH (Helicase)
XPF, which cuts the backbone on the 5' side of the
damage
XPG, which cuts the backbone on the 3' side

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Some mutations in XPB and XPD also produce
signs of premature aging


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Transcription-Coupled repair
Protein: ERCC6
recognizes RNApol

Mutation in gene:
Cokayne Syndrom:
MR
Nerve disease
Sensibility to sun
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