DNA

Repair
Vipin Shankar
DNA Repair
• Damaged in numerous ways and
times.
• But the effect of damage is not seen
as frequently as it occurs.
• The mechanisms of DNA repair, is
highly developed in all organisms
from bacteria to humans.
• These repair mechanisms keep the
mutations at the somatic and the
germ line level at a tolerable level.
DNA repair: general over view
• All repair systems are composed
of one or more proteins.
• Most of the repair mechanisms
are multi-step processes.
– One or more protein in the system
detect an irregularity in the DNA
structure.
– The abnormality is removed by the
action of DNA repair enzymes.
How important is Repair?
• There is no single molecule
whose integrity is as vital to the
cell as the DNA.
• Necessary for hereditary
information.
• Blue print for the operation of
individual cells.
Types of DNA Damage
• Missing Bases:
– The glycosidic linkage between the
DNA bases and the deoxyribose
sugar (of purine nucleotides) is
spontaneously broken.
– This is called depurination.
– The loss of a base creates an a-
basic site : apurinic/apyrimidinic
site.
Types of DNA damage…
• Altered bases:
– Bases can be changed into
strikingly different compounds by
a variety of chemical and physical
agents.
– Ionizing radiations like β -
particles can break purine and
pyrimidine rings and cause several
types of alterations.
– The most frequent substitutions are
made in thymine.
Types of DNA damage…
• Thymine dimers
– DNA helix becomes distorted as
thymine residues are pulled
towards one another.
– This causes distortion.
– Due to distortion, hydrogen
bonding to adenine in the 2nd strand
is weakened.
– This causes inhibition of
advancement of replication fork.
Types of DNA damage…
• Single stranded breaks:
– A variety of agents can break the
phosphodiester bonds.
– Peroxides, sulphydril containing
compounds, metal ions, etc.
– Ionizing radiations can also
produce breaks.
Types of DNA damage…
• Double stranded breaks
– If a DNA molecule has sufficient
number of randomly located single
stranded breaks, two breaks may
be situated opposite one another,
resulting in breakage of double
helix.
– Caused by x-rays, γ-rays and
radiations.
Types of DNA damage…
• Cross linking
– Some antibiotics (mitomycin C),
reagents (Nitric ion) can form
covalent linkages between a base
in one strand and another base in
the complementary strand.
– This prevents strand separation
during replication.
Repair mechanisms
• Repair mechanisms can either
repair the damaged DNA or
• Sometimes the damage is not
removed but the mechanisms
only skirt around it and help the
cell to survive with the damage.
Different repair mechanisms
• Photoreactivation.
• Excision repair.
– Base excision repair.
– Nucleotide excision repair.
• Mismatch repair.
• Recombination repair.
• SOS – Error prone repair.
Photoreactivation
• Light depended repair mechanism.
• Carried out by enzyme DNA
photolyase.
• The enzyme binds to thymine dimers
in the dark.
• The enzyme is activated by light.
• On absorption of light, the enzyme
catalyses the cleavage of the cross
link.
• DNA photolyase can also cleave C-C
and C-T dimers.
Visible
light
Excision repair
• 3 step reaction.
– A DNA repair endonuclease, or
endonuclease containing enzyme
complex, binds to and excises the
damaged base or bases in the
DNA.
– A DNA polymerase fills in the gap
using the undamaged
complimentary strand as a
template.
– DNA ligase seals the break left by
the polymerase
Base excision repair
• Initiated by a group of enzymes called
DNA glycolases, which break the
glycosidic bond between the damaged
bases and its sugar.
• This leaves an a-basic site.
• The a-basic site is recognized by AP
endonucleases, which cut the DNA
strand on either side of the a-basic
site.
• DNA Pol-I fills the gap and DNA
ligase seals the nick.
Nucleotide excision repair
• Recognizes damaged regions based on
their abnormal structure as well as
their abnormal chemistry.
• Steps.
– Damage recognition.
– Binding of multi-protein complex to the
damaged site.
– Double incision of the damaged strand
several nucleotides away from the damage
on both sides.
– Removal of the damaged regions between
the nicks.
– Gap filling by DNA polymerase.
– Ligation.
NER in E. coli
• 3 proteins uvrA, uvrB & uvrC.
• 2 molecules of uvrA and one
molecule of uvrB form a
complex. This is an energy
dependent step.
• This complex then binds to DNA
at the damaged site.
• In some cases the complex binds
to undamaged DNA and then
slide on to the damaged regions.
NER in E. coli…
• On reaching the damaged region,
further ATP hydrolysis results in uvrA
dissociation.
• uvrC now binds to uvrB.
• Binding of uvrC triggers uvrB to nick
the DNA 4nts to the 3’ end of the
damage site.
• This in turn triggers uvrC to nick the
DNA 7nts 5’ to the damage site.
• Another protein uvrD also called
Helicase-II, with the hydrolysis of
ATP, displace the damage containing
nucleotide.
NER in E. coli…
• uvrB is then displaced.
• DNA Pol-I fills the gap and
DNA ligase seals the nick.
Mismatch Repair
• The structure of DNA obeys the
A-t/G-C rule.
• Incorrect nucleotides are
sometimes added to the growing
DNA strand by mistake, during
replication.
• These mistakes are usually
corrected by the proof reading
mechanism of DNA polymerase.
• But at times, these mechanisms
fail to remove these mismatches.
Mismatch repair…
• The methyl directed mismatch
repair system in E. coli has been
extensively studied.
• The daughter strand and the
parent strand are distinguished
by the methylation present on
the parent strand.
• Three proteins, Mut L, Mut S &
Mut H detect and direct the
removal of the mismatches.
Mismatch repair…
• Mut H distinguishes between the
parental strand and the daughter
strand.
• Mut S locates the mismatch in the
DNA and forms a complex with Mut
L which is a linking protein.
• The Mut S – Mut L complex binds
with Mut H by means of DNA
looping.
• This stimulates Mut H to make a cut
in the non-methylated strand.
Mismatch repair…
• After the DNA strand is cut, an
exonuclease digests the non-
methylated strand in the
direction of the mismatch and
proceed just beyond the
mismatch.
• The gap in the daughter strand is
repaired by DNA Pol – I and
DNA ligase.
Recombination repair
• Also called post-replication repair.
• Requires the completion of repair.
• In strands with pyrimidine dimers,
replication is halted at the dimer
regions.
• But, after a halt, the replication
continues at a slower pace, leaving a
gap in the daughter strand at the dimer
region.
• Recombination occurs between the
gapped strand and its homologue on
the other daughter duplex.
Recombination repair…
• Since, the second duplex has no
dimer, the gap is filled by DNA
polymerase and ligase.
• The DNA damage still exists in
one strand, but the cell has at
least managed to replicate DNA.
• Sooner or later other repair
mechanisms will act on the
damage.
SOS – Error prone repair
• Induced by DNA damage,
including UV induced damage.
• Depends on the products of rec
A gene; rec A co-protease.
• rec A co-protease cleaves lex A
protein.
• The cleaved lex A, fails to
repress the expression of Umu C
and Umu D.
SOS- Error prone repair…
• These proteins foils the proof
reading ability of replication.
• Induces the inclusion of non-
complimentary base pairs at the
damage region.
• The error remains, but the cell
maintains to replicate its DNA.
• So called the Error-Prone
mechanism.