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DNA repair mechanism

DNA repair refers

to a collection of DNA damage
resulting in
processes by multiple
which a cell broken
identifies and
corrects damage
to the DNA
molecules that
encode its
Hoeijmakers, 2001
Types of DNA Damage
Deamination: (C U and A hypoxanthine)
Depurination: purine base (A or G) lost
T-T and T-C dimers: bases become cross- linked, T-
T more prominent, caused by UV light (UV-C
(<280 nm) and UV-B (280-320 nm)
Alkylation: an alkyl group (e.g., CH3) gets added to
bases; chemical induced; some harmless, some
cause mutations by mispairing during
replication or stop polymerase altogether
Types of DNA Damage (cont.)

5. Oxidative damage: guanine oxidizes to 8-oxo-guanine,

also cause SS and DS breaks, very important for
6. Replication errors: wrong nucleotide (or modified nt)
7. Double-strand breaks (DSB): induced by ionizing
radiation, transposons, topoisomerases, homing
endonucleases, mechanical stress on
chromosomes, or a single-strand nick in a single-
stranded region (e.g., during replication and
Types of DNA Damage (cont.)

Damage caused by exogenous agents

UV-B light causes cross linking between
adjacent cytosine and thymine bases
creating pyrimidine dimers. This is called
direct DNA damage
radicals is called indirect DNA damage.
Types of DNA Damage (cont.)
Ionizing radiation such as that created by radioactive
decay or in cosmic rays causes breaks in DNA
Thermal disruption at elevated temperature increases
the rate of depurination (loss of purine bases from the
DNA backbone) and single strand breaks. For
example, hydrolytic depurination is seen in the
thermophilic bacteria, which grow in hot springs at 85
250 C.[5] The rate of depurination (300 purine
residues per genome per generation) is too high in
these species to be repaired by normal repair
machinery, hence a possibility of an adaptive response
cannot be ruled out.
Types of DNA Damage (cont.)

Industrial chemicals such as vinyl

chloride and hydrogen peroxide, and
environmental chemicals such as
polycyclic hydrocarbons found in smoke,
soot and tar create a huge diversity of
DNA adducts- ethenobases, oxidized
bases, alkylated phosphotriesters and
Cross linking of DNA just to name a few.

The most common lesion that occurs in DNA is


Molecules in the gene do undergo major changes due to

thermal fluctuations. About 5,000 purine bases (adenine and
guanine) are lost per day from the DNA of each human cell
because of the thermal disruption of their N-glycosyl linkages
to deoxyribose (depurination)
Alkylation of DNA by alkylating agents
Repair mechanism

Cells are known to eliminate three types

of damage to their DNA by chemically
reversing it. These mechanisms do not
require a template, since the types of
damage they counteract can only occur
in one of the four bases.
1. The formation of thymine dimers (a common
type of cyclobutyl dimer) upon irradiation with
UV light results in an abnormal covalent bond
between adjacent thymidine bases. The
photo reactivation process directly reverses
this damage by the action of the enzyme
photolyase, whose activation is obligately
dependent on energy absorbed from blue/UV
light (300500 nm wavelength) to promote
Another type of damage, methylation of guanine
bases, is directly reversed by the protein methyl
guanine methyl transferase (MGMT), the bacterial
equivalent of which is called ogt. This is an expensive
process because each MGMT molecule can only be
used once; that is, the reaction is stoichiometric rather
than catalytic.[12] A generalized response to
methylating agents in bacteria is known as the
adaptive response and confers a level of resistance to
alkylating agents upon sustained exposure by
upregulation of alkylation repair enzymes.[13]
The third type of DNA damage reversed
by cells is certain methylation of the
bases cytosine and adenine.
1. When only one of the two strands of a
double helix has a defect, the other strand
can be used as a template to guide the
correction of the damaged strand. In order to
repair damage to one of the two paired
molecules of DNA, there exist a number of
excision repair mechanisms that remove the
damaged nucleotide and replace it with an
undamaged nucleotide complementary to
that found in the undamaged DNA strand.
Base excision repair (BER), which repairs damage to a
single base caused by oxidation, alkylation, hydrolysis,
or deamination. The damaged base is removed by a
DNA glycosylase, resynthesized by a DNA polymerase,
and a DNA ligase performs the final nick-sealing step.
Nucleotide excision repair (NER), which recognizes
bulky, helix-distorting lesions such as pyrimidine dimers
and . A specialized form of NER known as transcription-
coupled repair deploys NER enzymes to genes that are
being actively transcribed.
Mismatch repair (MMR), which corrects errors of DNA
replication and recombination that result in mispaired
(but undamaged) nucleotides
Deaminated C

Base Excision Repair (BER)

Variety of DNA glycosylases,
for different types of damaged

AP endonuclease recognizes
sites with a missing base;
cleaves sugar-phosphate

phosphodiesterase removes
the sugar-phosphate lacking
the base.
Pyrimidine dimer- Two adjacent
pyrimidine residues in the same
strand of DNA, which have
become covalently cross-linked
(e.g. by UV radiation)

The first step is cleavage of the

phosphodiester backbone next
to the distortion.

The second step is excision of

the lesion and resynthesis of
DNA in its place
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
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
Nucleotide-Excision Repair in E. coli and Humans
Excision and Restoration

reaction: two

A filling by
polymerase of
the gap
created by

The sealing by
DNA ligase of
a nick left in
the repaired
A double-strand break (DSBs) occurs in
one of the paired DNAs followed by
enzymatic trimming back of nucleotides on
the new single-strand ends.
A free 3' end invades the unbroken helix
and displaces a loop of single strand DNA.
A DNA polymerase elongates the free 3'
end of the invading strand, further
displacing the looped out strand, which
then pairs with an exposed single-strand
on the opposing helix.
The displaced strand serves as a template
for enzymatic extension from the 3' end of
the paired single strand, which eventually
crosses the junction and switches
templates. As 3' and 5' ends meet, strands
join to form two Holliday junctions.
There are two ways to resolve each
Holliday junction by single cleavage and
rejoining, so there are four ways to resolve
the double Holliday structure by two
cleavages and rejoining
Double-strand breaks, in which both
strands in the double helix are severed,
are particularly hazardous to the cell
because they can lead to genome
rearrangements. Three mechanisms exist
to repair DSBs: non-homologous end
joining (NHEJ), micro homology-mediated
end joining (MMEJ) and homologous
In NHEJ, , a specialized DNA ligase that forms a complex
with the cofactor XRCC4, directly joins the two ends.To
guide accurate repair, NHEJ relies on short homologous
sequences called micro homologies present on the
single-stranded tails of the DNA ends to be joined. If
these overhangs are compatible, repair is usually
NHEJ can also introduce mutations during repair. Loss
of damaged nucleotides at the break site can lead to
deletions, and joining of nonmatching termini forms
translocations. NHEJ is especially important before the
cell has replicated its DNA, since there is no template
available for repair by homologous recombination.
DNA ligase, shown
above repairing
chromosomal damage,
is an enzyme that joins
broken nucleotides
together by catalyzing
the formation of an
internucleotide ester
bond between the
phosphate backbone
and the deoxyribose
Homologous recombination requires the
presence of an identical or nearly
identical sequence to be used as a
template for repair of the break. The
enzymatic machinery responsible for this
repair process is nearly identical to the
machinery responsible for chromosomal
crossover during meiosis.
. This pathway allows a damaged
chromosome to be repaired using a sister
chromatid (available in G2 after DNA
replication) or a homologous chromosome as
a template. DSBs caused by the replication
machinery attempting to synthesize across a
single-strand break or unrepaired lesion cause
collapse of the replication fork and are typically
repaired by recombination.
Topoisomerases introduce both single- and
double-strand breaks in the course of
changing the DNA's state of supercoiling,
which is especially common in regions near an
open replication fork. Such breaks are not
considered DNA damage because they are a
natural intermediate in the topoisomerase
biochemical mechanism and are immediately
repaired by the enzymes that created them
DNA damage checkpoints
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. DNA
damage checkpoints occur at the G1/S and G2/M boundaries. An
intra-S checkpoint also exists. Checkpoint activation is controlled
by two master kinases, ATM and ATR. ATM responds to DNA
double-strand breaks and disruptions in chromatin structure,
whereas ATR primarily responds to stalled replication forks.

These kinases phosphorylate downstream targets in a signal

transduction cascade, eventually leading to cell cycle arrest. A
class of checkpoint mediator proteins including BRCA1, MDC1,
and has also been identified. These proteins seem to be required
for transmitting the checkpoint activation signal to downstream
The SOS response

The SOS response is the term used to

describe changes in gene expression in
Escherichia coli and other bacteria in
response to extensive DNA damage.
SOS repair occurs when cells are
overwhelmed by UV damage - this
allows the cell to survive but at the cost
of mutagenesis.
The SOS response

SOS response only triggered when other

repair systems are overwhelmed by
amount of damage so that unrepaired
DNA accumulates in the cell.
The error-prone repair mechanism
involves DNA pol. III and 2 other gene
products encoded by UmuCD.
The SOS response

The UmuCD proteins are produced in

times of dire emergency and instruct
DNA pol. III to insert any bases opposite
the thymine dimers, as the DNA damage
would otherwise be lethal.

The risk of several mutations is worth the

risk as measured against threat of death.
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:
sfiA (sulA) - a cell division inhibitor (repair
UmuCD, 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

recA & lexA
SOS boxes are 20-nucleotide long sequences
near promoters with palindromic structure and
a high degree of sequence conservation. This
distinction in promoter sequences causes
differential binding of LexA to different
promoters and allows for timing of the SOS
response. Logically, the lesion repair genes
are induced at the beginning of SOS
Once the DNA damage is repaired or
bypassed using polymerases or through
recombination, the amount of single-
stranded DNA in cells is decreased,
lowering the amounts of RecA filaments
decreases cleavage activity of LexA
homodimer which subsequently binds to
the SOS boxes near promoters and
restores normal gene expression.
Pathological effects of poor DNA
Experimental animals with genetic
deficiencies in DNA repair often show
decreased lifespan and increased cancer
DNA repair rate is an important
determinant of cell pathology
Hereditary DNA repair disorders

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
Hereditary DNA repair disorders

Werner's syndrome: premature aging

and retarded growth
Bloom's syndrome: sunlight
hypersensitivity, high incidence of
malignancies (especially leukemia).
ataxia telangiectasia: sensitivity to
ionizing radiation and some chemical
Hereditary DNA repair disorders

progerias" ("accelerated aging

diseases") because their victims appear
elderly and suffer from aging-related
diseases at an abnormally young age,
while not manifesting all the symptoms of
old age.
Other diseases associated with reduced
DNA repair function include Fanconi's
anemia, hereditary breast cancer and
hereditary colon cancer.
Case study: DNA repair UV-
irradiated repair-mechanism
Case study: DNA repair UV-
irradiated repair-mechanism
Materials and Apparatus
Stock culture of Escherichia coli from biological supply
Nutrient broth medium (sterile): peptone/beef
Nutrient agar medium (sterile): peptone/beef
Agar plates with lids
Sterile cotton
UV source (15 watt, in fluorescent desk lamp)
UV goggles or sunglasses approved for blocking UV)
Transfer loops
Flame source: Bunsen burner or alcohol lamp
Incubator, set to 38C
Stop watch
Marking pencils
Case study: DNA repair UV-
irradiated repair-mechanism
Day 1 Procedure
Mass 1.0 grams of a soil sample to be used as a source of
microbial decomposers.
Label one agar plate on the BOTTOM with your initials and the
date Inoculate the agar with a sample from the stock E. coli
Wearing approved safety goggles for protection against
ultraviolet radiation place the inoculated plate under the UV
light at a vertical distance of 15 cm from the light source and
uncover the plate. Turn on the light for 60 seconds.
Turn off the light; replace the cover on the agar plate.
Incubate the plate for 24 hours at 38C in minimal medium.
Day 2 Procedure
After 24 hours, remove the plate from the incubator and
examine for signs of bacterial growth.
Count and record the number of bacterial colonies.