1. MECHANISMS OF DNA
REPAIR
Dr Radhouane GOUIAA
Policlinique CNSS
3018 Sfax – TUNISIA
5th
ADVANCED POSTGRADUATE COURSE
2nd
Training Session
June 2003

2. DNA DAMAGE
1- Tautomerisation: spontaneous
Guanine: Keto form: pairs with Cytosine
Enol form: mispairs to Thymine
Thymine: Keto and Enol forms
Adenine and Cytosine: Amino and Imino forms

3. 1- Tautomerisation:
2- Deamination:
spontaneous base degradation
Base Pairing Deamination Mispairing
C G U A
A T Hypoxanthine C
G C Xanthine C

4. 3- Oxidation:damaging bases by free
radicals of oxygen
4- Alkylation: addition of alkyl groups to
bases or backbone DNA
5-Depurination: loss of Adenine or
Guanine bases: 10 000 bases per day per
cell
Other DNA hydrolysis reactions
6-Pyrimidine dimers: by sunlight
( UV ).
7-Single or double strand break.

5. DNA REPAIR
MECHANISMS
Three kinds:
A-Damage reversal
B-Damage removal
C-Damage tolerance

6. A-Damage reversal
may be divided in three different kinds:
1- Photo reactivation: most simple way
for DNA repair : a single step reaction.
Photolyase enzyme can split pyrimidine dimers:
breaks the covalent bond
Existence in mammalian not yet proved.

7. A-Damage reversal
1- Photo reactivation:
2-Most of damaged bases are repaired by
one enzyme, removes mismatched base restoring
the correct one. Without the need to break the DNA
backbone:
- Uracil, product of cytosine deamination, is
detected, removed by uracil N-glycosylase, and
replaiced by cytosine.

8. -Hypoxanthine, product of adenine deamination, is
recognized, removed by hypoxanthine N-
glycosylase then replaced by adenine
-Alkylation is repaired by enzymatic transfer of
methyl group by Methyl Guanine-DNA
MethylTransferase ( M G M T ), and we will have
Guanine
-Demethylation is used to repair 1-methyl adenine
and 3-methyl cytosine.

9. A-Damage reversal
1- Photo reactivation:
2-Most of damaged bases
3-Ligation of simple strand breaks:
Simple breaks in one strand are repaired by DNA
ligase.

10. A-Damage reversal
B-Damage removal:
Is the most common repair mechanism. It includes:
base excision repair, nucleotide excision repair, and
mismatch repair.

11. 1-Base excision repair ( B E R ):
It repairs small, non bulky DNA lesions: methylated,
oxidized, reduced bases. It is estimated to occur 20
000 times a day in each cell in our body:damaged
or inappropriate base is removed from its sugar
linkage and replaced.

12. Different steps of BER:
a- removal of the damaged base by a DNA
glycosylase. Eight enzymes,each one
responsible for identifying and removing a
specific kind of base damage.
b- removal of its deoxyribose phosphate in the
backbone, producing a gap: an AP site.Two
genes encoding enzymes with this function.

13. Different steps of BER:
c- replacement with the correct nucleotide.
Done by DNA polymerase beta ( one of at
least 11 DNA polymerases encoded by our
genes ), using the other strand as a template.
d- ligation of the break in the strand. Two
enzymes are known that can do this.

14. 1-Base excision repair ( B E R ):
2-Nucleotide excision repair ( N E R ):
The process of NER is biochemically complicated,
30 distinct proteins that function as a large complex
called the nucleotide excision repairosome.
- The most important DNA repair pathway,
- The sole repair system for bulky DNA lesions,
which creates a block to DNA replication and
transcription.
- Can also repair many of the same defects that are
corrected by direct repair, base excision and
mismatch repair

15. ( N E R ):
It probably recognizes lesions that distort the DNA
Steps in NER are:
a- Recognition of damage by one or more protein
factors.
b- Assembly of repair complex: nucleotide excision
repairosome.
c- Double incision of the damaged strand several
nucleotides away from the damaged site, on both
sides, by an endonuclease.

16. ( N E R ):
d- Removal of the short segment ( about 24 to 32
nucleotides ) containing the damaged region, by an
exonuclease.
e- Filling in of the resulting gap by a DNA
polymerase: synthesizes DNA using the opposite
strand as a template.
f- Ligation: a DNA ligase binds the synthesized
piece into the backbone.

17. 1-Base excision repair ( B E R ):
2-Nucleotide excision repair ( N E R ):
3- Mismatch repair ( M M R ):
This process occurs after DNA replication as a last
"spellcheck" on its accuracy.

18. ( M M R ):
Incorrect bases incorporated as a result of
mistakes during DNA replication ( base mispairs,
short insertions and deletions ) are excised as
single nucleotides by a group of repair proteins
which can scan DNA and look for incorrectly paired
bases (or unpaired bases) which will have aberrant
dimensions in the double helix.
Synthesis of the repair patch is done by a DNA
polymerase .

19. A-Damage reversal
B-Damage removal
C-Damage tolerance
It is not truly repair but a way of coping with
damage:

20. C-Damage tolerance:
1- Double strand break ( D S B ):
Naturally occurring reactive oxygen molecules and
ionizing radiation are prevalent sources of such
damage.
DSB’s are a major cytotoxic lesion : even a single
unrepaired DSB can be a lethal event.
There are two different mechanisms of repair:

21. 1- Double strand break:
a- End joining repair of DSB’s:
Joins broken chromosome ends in a manner that
does not depend on sequence homology and may
not be error free. incorrect ends may be joined, and
repair mechanism causes sequence errors.

22. a- End joining repair of DSB’s:
Three steps in end joining repair of DSB's:
1- Recognition of broken ends.
2- Unwinding of short stretch of DNA to uncover
short regions of homology "microhomologies"
3- Removal of unpaired regions and ligation of
products.
This mechanism: called non homologous end-
joining.

23. a- End joining repair of DSB’s:
b- Recombination repair: homologous
recombination:
It is an important and preferred mechanism of repair
since it is least likely to result in mutations:
broken ends are repaired using the information on
the intact homologous chromosome.
Requires an extensive region of sequence
homology between the damaged and template
strands.

24. C-Damage tolerance:
1- Double strand break ( D S B ):
a- End joining repair of DSB’s:
b- Recombination repair:
2- Error prone repair:
It is used when all else fails + + +

25. 2- Error prone repair:
Involves the replication machinery bypassing sites
of base damage, allowing normal DNA replication
and gene expression to proceed downstream of the
(unrepaired) damage.
Involves low-fidelity DNA polymerases that are able
to bypass DNA lesions that stall the high-fidelity
polymerases required for DNA replication.

26. 2- Error prone repair:
To overcome the block, these 'sloppy copiers'
add nucleotides to the replicating strand
opposing the DNA lesion,
-allowing replication to continue,
-and introducing mutations into the newly
synthesized sequence.

27. SEQUENTIAL EXPRESSION OF
REPAIR SYSTEMS:
- Damage reversal
- Excision-resynthesis ( E B R, then N E R, then
M M R )
- Recombination
- And finally error prone repair.

28. DNA DAMAGE, DNA REPAIR,
AND AGING:
DNA damage, particularly oxidative lesions, is
thought to contribute to aging. In fact, some
scientists believe : accumulation of uncorrected
DNA damage over years is a major cause of aging.

29. following observations:
* Animals with fastest rates of DNA repair have
longest life spans.
* Animals with highest rates of oxidative damage by
free radicals generally have shortest life spans.
* In lower life forms, anti-oxidant supplements, which
can correct and prevent DNA damage, increase life
span.
* Exposure to external causes of DNA damage
decreases life span.
* Humans who have genetic diseases resulting in
greater spontaneous DNA damage or inefficient DNA
repair often show signs of premature aging.

30. PREVENTING DNA DAMAGE AND
IMPROVING THE CAPACITY OF DNA
REPAIR:
These two goals seem possible by:
1- insuring enough intake of antioxidants as
vitamins C and E and beta-carotene.
2- caloric restriction ( reduction of total daily
calorie intake by about 35% for animals ).
3- avoiding exposure to UV and ionizing
radiations, and toxic chemicals.
4- fighting infections

31. CONCLUSION
DNA:many kinds of damage: base damage,
mispairing, single or double strand break
Many repair mechanisms:
- Damage reversal (Photoreactivation, ligation…)
- Damage removal: BER, NER, and MMR
- Damage tolerance: end joining repair,
recombination repair, and error prone repair.
These mechanisms are not always error free
DNA damage contribute to aging
Preventing DNA damage and improving capacity
of DNA repair may delay aging.

32. THANK YOU

33. REFERENCES
1) Troen BR. The biology of aging. Mt Sinai J
Med 2003 Jan;70(1):3-22.
2) de Boer J. and al. Premature aging in mice
deficient in DNA repair and transcription.
Science 2002 May 17;296(5571):1276-9.
3) Woods C G. DNA repair disorders. Arch Dis
Child 1998;78:178-184.
4) Friedberg E C. DNA damage and repair.
Nature 2003;421:436-440.
5) Wood R D. and al. Human DNA repair
genes. Science 2001 Feb 16;291:1284-1289.