3. Introduction
DNA repair refers to a collection of processes by which a
cell identifies and corrects damage to the DNA
molecules that encode its genome.
Sources of damage:
1-loss of a bases resulting in apurinic/apyrimidinic (AP)
sites (abasic sites).
2-base modifications, such as alkylations or
deamidations which converts cytosine, adenine and
guanine to uracil.
4. 3-Replication errors and base conversions can generate
mismatch nucleotide pairs
4-Failures in normal DNA metabolism by
topoisomerases and nuclease or ionizing radiation can
generate single-strand and double-strand breaks .
5-Photodamage by uv light can generate pyrimidine
dimers, such cyclobutane pyrimidine dimers (CPDs)
Chemical agents and reactive oxygen species (ROS) can
modify bases .
5. N.Bs:
Majority of DNA damage affect the 1ry structure of
double helix .
DNA repair is dependent on many factors :
-cell type - age of the cell -extracellular environment
A cell that has accumulated a large amount of DNA
damage can enter one of three possible states:
1-an irreversible state of dormancy, known as senescence
2-cell suicide, also known as apoptosis
3-unregulated cell division, which can lead to cancer
6. DNA Damage & Mutation
DAMAGE MUTATION
1-Damages are physical 1-A mutation is a change in the
abnormalities in the DNA base sequence of the DNA.
2-DNA damages can be 2-A mutation cannot be
recognized by recognized by enzymes once
enzymes, and, thus, they can the base change is present in
be correctly repaired. both DNA
3-If a cell retains DNA strands, and, thus, a mutation
damage, transcription of a cannot be repaired.
gene can be 3-Mutations can cause
prevented, and, thus, translat alterations in protein
ion into a protein will also be function and regulation.
blocked Mutations are replicated
when the cell replicates
7. REPAIR MECHANISMS
In addition to DNA polymerase 3’à5’ exonuclease(the
DNA Pol III has proofreading capabilities that
correct replication mistakes by means of
exonuclease activity working 3'->5') ..Mammalian
cells utilize TWO major DNA repair pathways:
- single strand damage -db strand breaks
Reverse Excision Non Homo Homo
BER NER MMR
8. A-SS damage : 1- reversal ( direct reversal ) :
These mechanisms do not require a template, since the
types of damage they counteract can occur in only one
of the four bases.(this type repaired without removing
abase or nucleotide)
E.X:
1-The formation of pyrimidine dimers upon irradiation
with UV light results in an abnormal covalent bond
between adjacent pyrimidine bases. The
photoreactivation process directly reverses this
damage by the action of the enzyme photolyase.
2- Another type of damage, methylation of guanine
bases, is directly reversed by the protein methyl
guanine methyl transferase (MGMT).
9. 2-Excision : In which the damaged base or bases are
removed and then replaced with the correct ones .
a-Base excision repair :
DNA's bases may be modified by deamination or
alkylation. the DNA glycosylase can recognize the
damaged site and remove its base forming AP site (
Apurinic/ Apyrimidinic). Then, the AP endonuclease
removes the AP site and neighboring nucleotides. The
gap is filled by DNA polymerase I and DNA ligase.
N.B: -Each DNA glycosylase is generally specific for one
type of lesion .
_ Humans have at least four types glycosylase with
different specifictices .
10.
11. B- Nucleotide excision repair :
NER differs from BER in several ways:
-It uses different enzymes.
-Even though there may be only a single "bad" base to
correct, its nucleotide is removed along with many
other adjacent nucleotides; that is, NER removes a
large "patch" around the damage .
- In NER a multisubunit enzyme hydrolyzes two
phosphodiester bonds one on either side of the
distorsion caused by lesion ( in human it hydrolyzes
the 6th bond on 3 side & the 22 bond on the 5 end
producing a fragment of 27-29 nucleotides ) resulting
in gap filled by DNA polymerase1 & finally DNA ligase
seals the nick .
12.
13. C- Mismatch repair :
To repair mismatched bases, the system has to know
which base is the correct one. In E. coli, this is
achieved by a special methylase called the "Dam
methylase", which can methylate all adenines that
occur within (5')GATC sequences. Immediately after
DNA replication, the template strand has been
methylated, but the newly synthesized strand is not
methylated yet. Thus, the template strand and the
new strand can be distinguished.
14.
15. B- Db strand damage : There are two mechanisms by
which the cell attempts to repair a complete break in a
DNA molecule:
1-Direct joining: of the broken ends. This requires
proteins that recognize and bind to the exposed ends
and bring them together for ligating. They would
prefer to see some complementary nucleotides but can
proceed without them so this type of joining is also
called Nonhomologous End-Joining (NHEJ).
Errors in direct joining may be a cause of the various
translocations that are associated with cancers.
16.
17. 2-Homologous Recombination:
. Here the broken ends are repaired using the
information on the intact sister chromatid (available
in G2 after chromosome duplication), or on the
homologous chromosome.
Two primary models:
1-DSBR pathway (sometimes called the double Holliday
junction model)
2- the synthesis-dependent strand annealing (SDSA)
pathway.
18.
19. N.Bs:
Whether homologous recombination or NHEJ is used to
the repair double-strand breaks is largely determined by
phase of cell cycle.
Homologous recombination repairs DNA before the cell
enters mitosis (M phase). It occurs during and shortly after
DNA replication, in the S and G2 phases of the cell
cycle, when sister chromatids are more easily available.
While NHEJ is predominant in the G1 phase of the cell
cycle, when the cell is growing but not yet ready to divide .
Cyclin-dependent kinases (CDKs), which modify the activity
of other proteins by adding phosphate groups to (that
is, phosphorylating) them, are important regulators of
homologous recombination in eukaryotes.
20. DNA damage check points
The global response to damage is an act directed
toward the cells' own preservation and triggers
multiple pathways of macromolecular repair, lesion
bypass, tolerance, or apoptosis (&the common features
of global response are induction of multiple genes, cell
cycle arrest, and inhibition of cell division ).
-After DNA damage, cell cycle checkpoints are activated
Checkpoint activation pauses the cell cycle and gives
the cell time to repair the damage before continuing to
divide .
21. - DNA damage checkpoints occur at the G1/S and G2/M
boundaries. Checkpoint activation is controlled by two
master kinases, ATM and ATR. ATM responds to DNA
double-strand breaks and disruptions in chromatin
structure,[31] whereas ATR primarily responds to stalled
replication forks. These kinases phosphorylate
downstream targets in a signal transduction cascade,
eventually leading to cell cycle arrest.
P53 is an important downstream target of ATM and -
ATR, as it is required for inducing apoptosis following
DNA damage.[33] At the G1/S checkpoint, p53 functions
by deactivating the CDK2/cyclin E complex.
Similarly, p21 mediates the G2/M checkpoint by
deactivating the CDK1/cyclin B complex .
22. -To get an idea of just the first layer of complexity in
these systems, let's assume there is some damage to
DNA, such as a single-strand break. Here is the process
that would happen:
1-DNA damage is detected by sensor proteins: PAR
activation this occurs within seconds of damage
detection. PARP 1 and PARP2 are activated by single
strand breaks and double strand breaks.
2-ATM activation (ATM and ATR kinases as seem to
send out the distress signal to recruit the right DNA
repair proteins. They do this by phosphorylating
mediator proteins.)
3-MDCI recruitment (This induces a signaling cascade.)
23. 4-RNF8 recruitment
5-RNFi68 recruitment
6-BRCA1 and 53BP1 recruitment
(Numbers 4-6 are particular proteins that are recruited
in a very specific order to do specific activities to repair
a single strand break.)
7-Based on certain factors such as timing and order of
recruitment, any one of these results may happen: the
cell cycle could be delayed, DNA could be repaired by
replacing nucleotide bases, differentiation may be
halted (senescence), cell death
(apoptosis), transcription and splicing controls, or
metabolic regulation.
24. Hereditary DNA repair disorders
Defects in the NER mechanism are responsible for
several genetic disorders, including:
Xeroderma pigmentosum: hypersensitivity to
sunlight/UV, resulting in increased skin cancer
incidence and premature aging
Cockayne syndrome: hypersensitivity to UV and
chemical agents
Trichothiodystrophy: sensitive skin, brittle hair and nails
Mental retardation often accompanies the latter two
disorders, suggesting increased vulnerability of
developmental neurons.
25. Other DNA repair disorders include:
Werner's syndrome: premature aging and retarded
growth
Bloom's syndrome: sunlight hypersensitivity, high
incidence of malignancies (especially leukemias).
Ataxia telangiectasia: sensitivity to ionizing radiation
and some chemical agents .
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