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DNA Repair Mechanisms
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DNA Repair - DNA is constantly being subjected to environmental interaction that causes the alteration or
removal of nucleotide bases. Radiations, chemical mutagens, heat, enzymatic errors, and spontaneous decay
constantly damage DNA.
If the damage is not repaired, a permanent mutation may be, introduced that can result in a number of
deleterious effects including loss of control over the proliferation of the mutated cell. In the long
evolutionary challenge to minimize mutation, cells have evolved numerous mechanisms to repair damaged
or incorrectly replicated DNA.
Many enzymes, acting alone or in concert with other enzymes, repair DNA. Most of the repair enzymes are
involved in recognizing the lesion, excising the damaged section of the DNA strand and using the sister
strand as a template, filling the gap left by the excision of the abnormal DNA.
Repair systems are generally placed in four broad categories: damage reversal, excision repair, double
strand break repair and post-replicative repair. Enzymes of the repair system have been conserved during
evolution. That is, enzymes found in E coli have homologues in yeast, fruitflies, etc.
Group of Enzymes Involved in Different Repair Mechanisms
Repair system
Base excision
Nucleotide Excision
Mismatch
Enzymes/Proteins
DNA glycosylase
AP endonuclease
DNA polymerase
DNA ligase.
UvrA, UvrB, UvrC
DNA polymerase I
DNA ligase.
Dam methylase
MutS, MutL, MutH
Exonuclease
DNA helicase
SSB proteins
DNA polymerase II
DNA ligase.
Mechanism of DNA Repair - Mechanisms for the repair of damaged DNA are probably universal. E coli
possesses at least three
different mechanisms for the
repair of DNA.
Direct Repair - Direct repair
involves neither removal nor
replacement of bases or
nucleotides; rather the
covalent modification in
DNA is simply reversed. It
may be of the following
types.
DNA Repair Mechanisms
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Damaged Reversal OR Photo Reactivation -Exposure of a cell to ultraviolet light can result in the
covalent joining of two adjacent pyrimidines producing a dimer. Although cytosine-cytosine, cytosine
thymine dimers are also formed, the principal products of UV irradiation are thymine thymine dimers.
These thymine dimers prevent DNA polymerase from replicating the DNA strand beyond the site of dimer
formation. In E coli, an enzyme called DNA photolyase (deoxyribodipyrimidine photolyase or photo
reactivating enzyme) detects and binds to the damaged DNA site.
Then the enzyme absorbs energy from visible light, which activates it so it can break the bonds holding the
pyrimidine dimer together. The enzyme then falls free of the DNA. This enzyme thus reverses the UV
induced dimerization. Photo reactivation or photo-restoration is a light dependent DNA repair mechanism in
which certain types of pyrimidine dimers are cleaved.
This repair pathway is found in many prokaryotes and lower eukaryotes but absent in higher eukaryotes.
Photo reactivation should not be confused with other, non enzymatic mechanisms of monomerization.
Direct Photo Reversal
This occurs during continued UV irradiation of DNA and reflects the establishment of equilibrium between
the monomer and dimer states of adjacent pyrimidines. Sensitized photo reversal occurs in the presence of
tryptophan, which donates an electron to facilitate monomerization.
ExcisionRepair - The percentage of mutations that can be handled by direct reversal is necessarily small.
Most mutations involving pyrimidine dimers are handled by a different mechanism.
Most of them are removed by a process called excision repair. Excision repair refers to the general
mechanism of DNA repair that works by removing the damaged portion of a DNA molecule.
During excision repair, bases or nucleotides are removed from the damaged strand. The gap is then patched
using complementarity with the remaining strand. This occurs by one of the two mechanisms-base excision
repair or nucleotide excision repair.
Base Excision Repair - Certain mutations are recognized by an enzyme called DNA glycosylases which
breaks the N glycosidic bond between the damaged base and its sugar ring in the DNA backbone. DNA
glycosylase can recognize a single, specific type of damaged or inappropriate base in DNA.
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DNA Repair Mechanisms
Nucleotide Excision Repair - Base excision can repair only one base pair change whereas bulky base
damage including
thymine dimers
are removed by
nucleotide
excision repair. If
the dimerization is
not reversed by
the photo
reactivation
system, then
nucleotide
excision repair
comes into
picture. Two
copies of the
protein product of
the uvrA gene
combines with one
copy of the
product of the
uvrB gene to form
a uvrA2-uvrB
complex that
moves along the
DNA, looking for
damage.
When the complex
finds damage such
as a thymine
dimer, with
moderate, to large
distortion of the
DNA double
helix, the uvrA2 dimer dissociates, leaving the uvrB subunit alone.
This causes the DNA to bend and attract the protein product of the uvrC gene, uvrC. Binding of uvrC
protein causes a conformational change in the DNA following uvrB to nick the DNA four to five
nucleotides on the 3' side of the lesion. After the 3' nicking by uvrB, uvrC protein nicks 5 bp away, towards
the 5' end.
The binding of uvrC and nicking reactions require A TP binding but not hydrolysis. The three components,
uvrA, uvrB and uvrC, are together called the ABC exonuclease. The enzyme helicase- II, the product of the
uvrD gene, then removes the 12-13 nucleotides along with uvrC.
DNA polymerase I fills in the gap and in the process, evicts the uvrB and DNA ligase close to the remaining
DNA Repair Mechanisms
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nick. This is another relatively simple system designed to detect helix distortions and repair them like base
excision repair. Nucleotide excision repair is present in all organisms.
Post Replicative Repair -When DNA polymerase encounters damage in DNA, it cannot proceed. Instead it
gives a gap for replication and proceeds up to 800 bp without replicating. Then again it starts replicating
after synthesizing a primer by primosome. These gaps are then repaired by using one of the two
mechanisms. Originally several proteins were known to facilitate the replication of DNA with lesions.
They were believed to interact with the polymerase to make it capable of using damaged DNA as a
template. In E. coli, polymerase I can copy damaged DNA. Pol V is error free and can incorporate' A'
opposite to thymine dimers. But sometimes, Pol V does errors for unknown reasons, especially during
stress.
One possible reason for this is that the error prone polymerase may have developed by evolutionary
processes. They create mutations at a time when the cell might need variability. In the second mechanism, a
replication fork creates two DNA duplexes.
Thus an undamaged copy of the region with the lesion exists on the other daughter duplex. The repair takes
place at a gap created by the failure of DNA replication, the process is called post-replicative repair. The
RecA protein has two major properties.
First, it coats single stranded DNA and causes the coated single stranded DNA to invade double stranded
DNA while displacing the other strand of that double helix. RecA continues to move the single stranded
DNA along the double stranded DNA until a region of homology is found.
The RecA protein is responsible for filling a post replicative gap in newly replicated DNA with a strand
from the undamaged sister duplex. The gap filling process then completes both strands. The RecA protein is
responsible for the damaged single strand invading the sister duplex.
Endonuclease activity then frees the double helix containing the thymine dimer. DNA polymerase I and
DNA ligase return both daughter helices to the intact state. The thymine dimer still exists, but now its
duplex is intact. Another cell cycle is available for photo reactivation or excision repair to remove the dimer.
SOS Repair - In E. coli, it is the last mechanism (till now discovered) by which a cell can repair its
damaged DNA. If SOS repair fails in eukaryotes, then the cell commits suicide by apoptosis.SOS response
is an inducible response to DNA damage resulting in increased capacity for DNA repair, inhibition of cell
division and altered metabolism.
SOS response is mediated by the activation of about 20 SOS genes which are normally expressed at low
levels due to transcriptional repression by the LexA repressor, which binds to operator sequences (SOS
boxes) upstream of each gene. When DNA becomes extensively damaged, single-stranded regions are
exposed.
Single stranded DNA interacts with RecA to produce an activated complex termed RecA which facilitates
cleavage of the LexA repressor by increasing the rate of LexA autoproteolysis. The cleaved LexA protein
can no longer bind to DNA and the SOS genes are derepressed.
DNA Repair Mechanisms
When damaged DNA is no longer present in the cell, RecA becomes inactivated and no longer facilitates
LexA cleavage. A high level of un cleaved LexA rapidly accumulates in the cell from the existing LexA
mRNA pool and the lex A gene and other SOS genes are shut down.
Mismatch Repair - Mismatch or non-Watson-Crick base pairs in a DNA duplex can arise through
replication errors, through deamination of 5' methylcytosine in DNA to yield thymine or thorough
recombination between DNA segments that are not completely homologous.
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DNA Repair Mechanisms
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If DNA polymerase introduces an incorrect nucleotide, this error is corrected by mismatch repair. Mismatch
repair system scans newly replicated DNA or daughter strand and finds the mismatch errors or nucleotides.
It uses 3 proteins named Mut S, Mut H and Mut L for the repair. Mut S binds to the mismatch site and it
waits there. Then Mut Land Mut H bind creating a loop starting from the site GATC to GATC. This loop
formation is driven by the A TP.
Then Mut H, which is assumed as endonuclease cleaves at the GA TC site on both the sides of the
mismatch. Then Mut U unwinds the strand. Then DNA polymerase synthesizes the new strand and ligase
joins the strand.
Nucleotide Mismatch Repair in Eukaryotes - Nucleotide excision repair in eukaryotes involves a large
number of genes. The mechanisms of nucleotide excision repair appear to be quite similar in eukaryotes and
bacteria, with bimodal incision followed by excision and resynthesis.
The repair patch following nucleotide excision repair in humans is slightly longer than that in E.coli (30 nt)
and is confusingly termed long patch repair, to distinguish it from the 1-2 nucleotide short patch repair
which occurs in base excision repair. In eukaryotes, transcription and repair are linked especially for
nucleotide excision repair.
The basis of this link is the general transcription factor TFIIH, which forms an essential part of both the
basal transcriptional apparatus of the cell and the complex of repair proteins, the repairosome.
In the transcription complex, the TFIIH core is associated with a cyclin-dependent kinase complex, whereas
in repair, it is associated with other repair proteins. Hence, repair is preferentially directed to the transcribed
strand of actively transcribed gene (transcription coupled repair) and is more active when transcription is in
process (transcription dependent repair).
After DNA polymerase III skips past a lesion such as a thymine dimer, the cell fills in the gap without using
template information for complementarity. The site will contain mutations, but at least the integrity of the
double helix will be maintained.
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