DNA damage & repair

UV irradiation
When life evolved it is thought that UV light levels
were much higher than they are now.

UV light kills cells, and inactivates viruses.

2 macromolecules strongly absorb UV light

1. Nucleic acids
DNA (peak absorbance at 260 nm)

2. Protein (peak absorbance at 280 nm)

So which macromolecule is the most sensitive?

 UV survival curves
The UV survival curve for both mutant and wild-type
indicates that there are repair systems to deal with UV –
damaged induced DNA.

2 key observations:
UV-irradiated bacteria if exposed to visible light
showed an increased survival relative to those not
exposed to visible light – PHOTOREACTIVATION

UV-irradiated bacteria if held in non-nutrient buffer for

several hours in the dark, also showed enhanced
survival relative to controls which had not – LIQUID
HOLDING RECOVERY or DARK REPAIR
 Photoreactivation repair
The enhanced survival of UV-irradiated bacteria
following exposure visible light is now known to be due
to PHOTOLYASE, an enzyme that is encoded by
E. coli genes phrA and phrB.
This enzyme binds to pyrimidine dimers and uses
energy from visible light (370 nm) to split the dimers
apart.
Phr- mutants were defective at photoreactivation.
Similar enzymes are found in other bacteria, plants and
eukaryotes (but not present in man).
(from T.A.Brown.
Genetics a molecular
approach)
 Dark repair or light independent
mechanisms

3 mechanisms:

1. Excision repair – removal of damaged

DNA strand followed by DNA synthseis
2. Recombinational repair - using other
duplexes for repair.
3. SOS error-prone ‘repair’ – tolerance of
DNA damage
 Excision repair
In this form of repair the gene products of the E.
coli uvrA, uvrB and uvrC genes form an enzyme
complex that physically cuts out (excises the
damaged strand containing the pyrimidine
dimers.
An incision is made 8 nucleotides (nt) away for
the pyrimidine dimer on the 5’ side and 4 or 5 nt
on the 3’ side.. The damaged strand is removed
by uvrD, a helicase and then repaired by DNA
pol I and DNA ligase.
Is error-free.
 Excision Repair in E.coli
5’ TT 3’ Damage recognised
3’ 5’ by UvrABC, nicks
made on both sides of
5’ TT 3’ dimer
3’ 5’
TT
Dimer removed by
5’ 3’ UvrD, a helicase
3’ 5’

Gap filled by DNA

5’ 3’ pol I and the nick
3’ 5’
sealed by DNA
ligase
 Excision repair
The UvrABC complex is referred to as an exinuclease.
UvrAB proteins identify the bulky dimer lesion, UvrA
protein then leaves, and UvrC protein then binds to UvrB
protein and introduces the nicks on either side of the dimer.
In man there is a similar process carried out by 2 related
enzyme complexes: global excision repair and transcription
coupled repair.
Several human syndromes deficient in excision repair,
Xeroderma pigmentosum, Cockayne Syndrome, and are
characterised by extreme sensitivity to UV light (& skin
cancers)
 Excision Repair Enzymes
UvrABC endonuclease (helix distortions)
DNA glycosylase (damaged base)
AP endonuclease (missing base)
Uracil N-glycosylase (uracil in DNA)
 Base excision repair
NOT a major form
of repair of UV-
induced DNA damage,
but an important form
of DNA repair
generally.

(from T.A.Brown. Genetics a

molecular approach)
 Recombinational Repair
Thymine 3’
dimer 5’ Recombination is dependent
5’ RecA protein
3’
3’
5’ Thymine 3’
dimer 5’
Undamaged parental strand
5’
‘recombines’ into the gap
opposite the dimer, leaving 3’
a gap on the other parental 3’
strand. 5’
 Recombinational Repair
Thymine 3’
dimer 5’
5’
3’
3’
Thymine 3’
5’ 5’
dimer

The gap in the undamaged 5’

parental strand is filled by DNA 3’
pol I and ligase. The thymine 3’
dimer can now be repaired by 5’
excision repair
 Homologous
DNA recombination

RecA protein is
essential for
homologous
recombination

(from T.A.Brown.
Genetics a molecular
approach)
 Summary
Both the dark repair mechanisms and photo-
reactivation are very accurate and can deal with low
levels of DNA damage.

However, extensive damage levels to elevated levels

of excision and recombinational repair, and also the
activation of another repair system which is error-
prone (SOS) repair

This error –prone repair mechanism is a last resort to

ensure survival
 Inducible Error-Prone Repair
Thymine 3’
dimer 5’
5’
3’
3’
5’ Thymine 3’
dimer 5’
Under extreme conditions
DNA synthesis is extended 5’
through the dimer, and any 3’
nucleotide is incorporated
3’
:. ERROR PRONE.
5’
 The SOS response
In response to extensive genetic damage there is a regulatory
system that co-ordinates the bacterial cell response. This
results in the increased expression of >30 genes, involved
in DNA repair, these include:
recA - activator of SOS response, recombination
sfiA (sulA) - a cell division inhibitor (repair before
replication)
umuC, D - an error prone bypass of thymine dimers
(loss of fidelity in DNA replication)
uvrA,B,C,D - excision repair
The SOS response is regulated by two key genes:
recA & lexA
Lex A repressor binds to a site where
RNA polymerase binds, and thereby
prevents transcription of SOS genes

LexA binding site

SOS gene

-35 -10 mRNA

RNA pol. binding sites

 The SOS regulon
Greater than 30 genes are co-ordinately regulated by a
repressor protein, known as the LexA repressor.
This repressor protein binds to the –35 region of all the SOS
genes at specific sites.
The binding of LexA protein prevents expression of all the
‘SOS’ genes, of which the lexA gene is one.
In the presence of DNA damage the RecA protein is
‘activated’ such that it binds to LexA protein , and promotes
LexA protein to undergo autocleavage to an inactive form.
This results in expression of al the SOS genes (including the
lexA gene and the recA gene)
 The SOS regulon
After DNA damage is dealt with the RecA protein no
longer promotes the auto-cleavage of the LexA
repressor.

This restores the LexA regulation until the next time

there is extensive genetic damage.

Should not think of as an all or nothing response, there is

fine tuning.
Also not wholely a desperate response to extreme
damage, as excision repair and recombinational repair
are also elevated in this response.
 Mismatch repair
Not a form of repair for UV-induced DNA damage, but
a mechanism for further ensuring that replication is
accurate.

Defects in man are associated with hereditary colon

cancer.
 Replication mismatches can are
corrected by a mismatch repair system
Normal

Replication 5’-GATCG-3’
3’-CTAGC-5’
5’-GATCG-3’ 5’-GAGCG-3’ M
3’-CTAGC-5’ 3’-CTAGC-5’
Normal
M M
5’-GATCG-3’
3’-CTAGC-5’
M
M = methylation
on old strand
 Dam methylation
The bacterial can discriminate between old and new
replicated strands.

Old strands are methylated at adenine in -GATC-

sequences by an enzyme called the Dam methylase.

So the mismatch on the newly replicated strand is

preferentially excised.

Mismatch repair systems are present in human cells

and defects lead to colon cancer.