DNA REPAIR

Usha Middha
Lecturer, Dpt. of Biochemistry, MLACW

WHY DNA repair?

DNA is a very stable molecule.

Presence of a variety of toxic substances,
and exposure to UV or ionizing radiation
subjects it to numerous chemical insults that
excise or modify bases and alter sugar
phosphate groups.(alterations caused by
harmful chemical and radiation)
Base substitution during replication
The DNA fidelity of DNA replication is
dependent on DNA polymerases.

Evident DNA repair system

130 genes responsible for DNA repair

Many kinds of DNA repair system
DNA repair mechanism is chemically
similar in Eukaryotes and E.coli
The DNA repair systems do not operate
on single-stranded nucleic acids and
hence the viruses(HIV) have very high
rates of mutation.

Difference between DNA damage and

Mutation
DNA damage

DNA Mutation

DNA damage is a chemical

alteration

Mutation is a change in a base

pair

Base modifications caused by

alkylating agents

Change of A to T,G and C

Pyrimidine dimers caused by UV

radiation
DNA damage can lead to DNA
mutation then it is know as
genotoxic

What makes DNA a stable molecule

The sugar Phosphate backbone bond is

extremely stable

The C-C bond in the sugar is also extremely

strong and are resistant to chemical attack(but
not strong acid and high temperature)

The phosphodiester bond is less stable than c-c

bond and sugar phosphate bond

At alkaline pH RNA hydrolyses(due to

phosphodiester
linkage) into nucleotides
rapidly and probably that is the reason for DNA
to evolve as genetic department ( elimination)
At room temperature and pH (8-9)

Double helical structure provides protection

against chemical attack
The hydrophobic nature of rings of bases(allows
to stack on one another and reduces area of
chemical attack)
Charged groups on the bases can react with
chemical molecules and so have to be protected
Hydrogen bonds between the bases provide the
first line of defence by allowing the DNA to form
gigantic cluster with the bases at the center.

The possibility of water to react with the

bases is minimal
All chemical components which are
water soluble therefore can not reach
the bases.
The N glycosidic bond is very stable
except at extreme pH.
The redundancy of genetic material

Sites of chemical damage DNA

susceptible

IMPORTANT SLIDE

Types of DNA repair

Direct reversal of the damage

Excision repair
Mismatch repair
The SOS response
Double strand break repair

Experimental demonstration of repair in

prokaryotes

Bacterial sample was grown

at normal conditions

Samples were drawn at

intervals from a population
of bacteria irradiated by UV
light.

The samples were further

plated on nutrient agar

The colonies that are formed

are counted.

The proportion of cells able

to produce colonies grown
plotted as a function of
ultraviolet dose.

Similar cytosine and thyminecytosine dimers

are likewise formed but at lesser rates.
75% are thymine dimers and 25% of the uv
caused lesions are (6-4) photoproducts
The 6th carbon of one pyrimidine is linked to
the 4th carbon of adjacent pyrimidine.
All cyclobutane pyrimidine dimers (CPDs)
locally distort DNAs base-paired structure
such that it can be neither transcribed nor
replicated.

Formation of the most toxic and mutagenic

DNA lesion -Thyminethymine
cyclobutanepyrimidine dimer
and their photoreactivation by the enzyme
photolyase in the presence of light

Formation of the most toxic and mutagenic

DNA
lesion
-Thyminecytosine
pyrimidine
dimer
and
their
photoreactivation by the enzyme photolyase
in the presence of light

Pyrimidine dimers may be restored to their monomeric forms through the action of
light-absorbing enzymes named photoreactivating enzymes or DNA
photolyases

These are present in many prokaryotes and eukaryotes (including goldfish,

rattlesnakes, and marsupials, but not placental mammals).
These enzymes are 55- to 65-kD monomers

They bind to a pyrimidine dimer in DNA, in the dark.

A
noncovalently
bound
chromophore,
in
some
species
an
N5,N10methenyltetrahydrofolate (MTHF; and in others a 5-deazaflavin, then absorbs
300- to 500-nm light and transfers the excitation energy to a noncovalently bound
FADH, which in turn transfers an electron to the pyrimidine dimer, thereby splitting
it.

Finally, the resulting pyrimidine anion re-reduces the FADH and the now
unblemished DNA is released, thereby completing the catalytic cycle.

DNA photolyases bind either dsDNA or ssDNA with high affinity but without regard to
base sequence.

Alkyltransferases Dealkylate Alkylated Nucleotides

The exposure of DNA to alkylating

agents such as Nmethyl-N-nitro-Nnitrosoguanidine
(MNNG)
yields,
among
other
products,
O6alkylguanine residues.

The formation of these derivatives is

highly mutagenic because on replication,
they frequently cause the incorporation
of thymine instead of cytosine.

O6-Methylguanine and O6ethylguanine lesions of DNA in all

species tested are repaired by O6alkylguanine DNA alkyltransferase,
which directly transfers the offending
alkyl group to one of its own Cys residues.
Since it gets inactivated(dies) after this
reaction it called suicide enzyme
Therefore the repair process is expensive
cost one enzyme per reaction.

The E. coli O6-alkylguanineDNA alkyltransferase

activity occurs on the 178-residue C-terminal
segment of the 354-residue Ada protein (the
product of the ada gene).
Its X-ray structure determined by Eleanor Dodson
and Peter Moody, reveals, unexpectedly, that its
active site Cys residue, Cys 321, is buried inside
the protein.
Apparently, the protein must undergo a significant
conformational change on DNA binding in order to
effect the methyl transfer reaction.

Ada proteins 92-residue N-terminal segment has an

independent
function:
It
repairs
methyl
phosphotriesters in DNA (methylated phosphate
groups) by irreversibly transferring the offending
methyl group to its Cys 69.

The NMR structure of Adas N-terminal domain

determined by Gregory Verdine and Gerhard Wagner,
reveals that Cys 69, together with three other Cys
residues, tetrahedrally coordinates a Zn2 ion. This
presumably stabilizes the thiolate form of Cys 69 over
its thiol form, thereby facilitating its nucleophilic attack
on the methyl group.

Intact Ada protein that is methylated at

its Cys 69 binds to a specific DNA
sequence, which is located upstream of
the ada gene and several other genes
encoding DNA repair proteins, thereby
inducing their transcription. Evidently,
Ada also functions as a chemosensor of
methylation damage.

Base excision repair

Base excision repair pathway
(BER).
(a)
A
DNA
glycosylase
recognizes a damaged base
and cleaves between the base
and
deoxyribose
in
the
backbone.
(b)
An
AP
endonuclease
cleaves the phosphodiester
backbone near the AP site.
(c) DNA polymerase I initiates
repair synthesis from the free
3 OH at the nick, removing a
portion of the damaged strand
(with its 53 exonuclease
activity) and replacing it with
undamaged DNA.
(d) The nick remaining after

A DNA glycosylase initiates base excision repair

Examples of bases cleaved by

DNA glycosylases:
Uracil (deamination of C)
8-oxoG paired with C (oxidation of
G)
Adenine across from 8-oxoG
(misincorporation)
Thymine across from G (5-meC
deamination)
Alkyl-adenine (3-meA, 7-meG,
hypoxanthine)

Base excision pathway

A glycosylase acts by hydrolyzing the glycosidic bond

& then DNA polymerase and DNA ligase restore an
intact strand

If a damaged base is not removed by base

excision before DNA replication
A fail-safe system

Nucleotide excision repair

(NER)

Recognizes bulky lesions that block DNA replication

(i. e. lesions produced by carcinogens)--example,
UV pyrimidine photodimers
Common distortion in helix
Incision on both sides of lesion
Short patch of DNA excised, repaired by
repolymerization and ligation
In E. coli, mediated by UvrABCD
Many more proteins involved in eukaryotes
Can be coupled to transcription (TCR, transcription
coupled repair)
Defects in NER underlie Xeroderma pigmentosum

Xeroderma pigmentosum

Autosomal recessive mutations in several complementation

groups
Extreme sensitivity to sunlight
Predisposition to skin cancer (mean age of skin cancer = 8 yrs
vs. 60 for normal population)

Nucleotide excision repair

UvrA recognizes
bulky lesions
UvrB and
UvrC make cuts

Structural distortion = signa

Fig. Nucleotide excision repair (NER) of pyrimidine dimmer and

other damage-induced distortions of DNA

Nucleotide excision
repair
It takes two forms in case of eukaryotes
1. CG-NER( Global genome NER)
2. TC-NER (Transcription coupled NER)

Global genome NER

Transcription coupled NER

It is similar to that of global NER

Except for the absence of XPC
Instead of XPC it is RNA polymerase
which is identifying the lesion and stall
the process of replication
After the XPC identifies the lesion the
XPA recruits all the proteins required for
NER as in Global NER

SOS repair allows DNA chain growth

across the damaged segments at the
cost of fidelity of replication.
It is an error prone process; even
though intact DNA strands are formed,
the strands contain incorrect bases
This allows the polymerisation to
proceed further across the dimer.

The RecA protein binds tightly to ssDNA but very weakly

to dsDNA.
The distortion resulting from a pyrimidine dimer produces
a short stable single stranded region to which RecA binds.
When DNA polymerase III encounters a dimer site to
which RecA is bound, RecA interacts with the subunit
of the polymerase and inhibits the editing function and
allows replication fork to advance.
The presence of RecA at the dimer site inhibits editing
and causes the mispaired base to remain in the daughter
strand as a mutation.

lexA binds to operators repressing the

synthesis of proteins involved in the SOS
response

RecA is activated by binding to the ssDNA to stimulate

LexA self cleavage.

Consequent synthesis of the SOS proteins

Results in the repair of the DNA damage

When the DNA lesions have been eliminated

RecA ceases stimulating LexA,s auto proteolysis

The newly synthesised LexA can then function as a

repressor which permits the cells to return to normal
type.