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.

Mechanism of DNA Repair

Mechanisms for the repair of damaged DNA are probably universal. E coli possess at least three
different mechanisms for the repair of DNA.

1) Remove damaged region

2) Resynthesis DNA

3) Ligation
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.

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.

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.

Certain glycosylases may also introduce a nick 3' to the site of the damage through associated
AP lyase activity. DNA glycosylase acts upon single bases and has no specificity for larger or
more complex lesion involving multiple bases. Interestingly, glycosylases repair or remove the
nitrogen base pair by flipping them out of the interior of the double helix in a process called
base flipping. DNA glycosylases generate an AP site (a purine/a pyrimidine), i.e., a sugar without
nitrogen base.

DNA glycosylase has narrow substrate specificity and no cofactor requirement. Once an AP site
is generated, it is recognized by a second repair-specific enzyme termed as AP endonuclease,
which introduces a nick 5 ' to the AP site. In E. coli, there are two types of AP endonucleases.

1. Endonuclease III This enzyme is encoded by the Xth gene and is important for nicking at 5'
end.

2. Endonuclease IV This enzyme is coded by the nfgene and is involved in oxidative damage
repair.

Following the 5' incision to the AP site, a further enzyme deoxyribophospodiesterase (dRpase),
is required to hydrolyse the 5' residue resulting in a single nucleotide gap.

DNA polymerase may initiate repair synthesis from this gap, replacing the missing nucleotide
residue with a repair patch of one nucleotide. Finally the nick remaining after repair synthesis is
closed by DNA ligase.
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 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.

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.
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.
 Recombination

Genetic recombination may be regarded as a process of breakage and repair between two DNA
molecules. In eukaryotes, the process takes place early in meiosis after each molecule has
replicated, and with respect to genetic markers, it results in two molecules of the parental type
and two recombinants. Molecular models of recombination are quite complex because
heteroduplexes may or may not lead to recombination between flanking genetic markers.

The Holliday Model

In the Holliday model of recombination, each of the participating DNA duplexes undergoes a
symmetrically positioned single-stranded nick in strands of the same polarity. The nicked strands

unwind in the region of the nicks, switch pairing partners, and form a heteroduplex region in
each molecule. Ligation anchors the ends of the exchanged strands in place, and further
unwinding increases the length of the heteroduplex region (termed branch migration). Two more
nicks and rejoinings are necessary to resolve the structure in Figure.
As the configuration is drawn, these breaks can be in the inner strands, resulting in molecules
that are nonrecombinant for markers flanking the heteroduplex region, or they can be in the outer
strands, resulting in molecules that are recombinant for outside markers.
Of the two possibilities for resolution of the structure, one results in recombination of the outside
markers and the other does not. These outcomes are equally likely because there is no
topological difference between the inner strands and the outer strands. To understand why, note
that an end-for-end rotation of the lower duplex in Figure. This type of configuration is known as
a Holliday structure.

The Holliday Model of Genetic Recombination

This model of recombination was first proposed by Robin Holliday in 1964 and re-established by
David Dressler and Huntington Potter in 1976 who demonstrated that the proposed physical
intermediates existed.

The basic (simple) model

Align two homologous DNA molecules.

Nick the DNA at the same place on the two molecules.

This must happen in strands with the same polarity.

Exchange strands and ligate.

The intermediate that is formed is called a Holliday intermediate or Holliday structure. The
shape of this intermediate in vivo is similar to that of the greek letter chi, hence this is also called
a chi form.

Visualization of the next step is made easier if one molecule is now rotated through 180š with
respect to the other. This also helps to emphasize the chi-shape of the intermediate:

Resolve the structure.

There are two ways in which this can happen:

 If the same strands are cleaved a second time then the original two DNA
molecules are generated:
  If the other strands are cleaved, then recombinant molecules are
generated:

A more realistic model

The above model is too simple and does not explain a number of genetic results, including the
occurence of two different recombinant bacteriophage in a single plaque in the Meselson-Weigle
experiment.

These can be explained by modifying the model slightly. As before, two homologous DNA
molecules must be aligned and nicked at the same place. Following strand exchange the
intermediate Holliday structure is formed. At this stage a new step is introduced:

Branch migration.

Migration of the branch can occur over many nucleotides in either direction. The result is a
physical transfer of part of one of the strands of one molecule with that of the other:
Once again, visualization of the next step is made easier if one molecule is now rotated
through 180° with respect to the other.

Resolve the structure.

There are still two ways in which this can happen, however, the consequences are different:

 If the same strands are cleaved a second time then nonrecombinant DNA
molecules are generated but they each contain a region of heteroduplex
DNA that spans the region of branch migration:

 If the other strands are cleaved, then recombinant molecules are

generated as before, however, each will also contain a region of
heteroduplex DNA that spans the region of branch migration: