DNA damage and DNA repair

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DNA DAMAGE AND DNA REPAIR
Background
The history of the repair of damaged DNA can be traced to the mid-1930s.
Since then multiple DNA repair mechanisms, as well as other biological
responses to DNA damage, have been discovered and their regulation has been
studied. This article briefly recounts the early history of this field.
By the early 1940s it was becoming evident that agents that elicit mutational
changes (such as ionizing and UV radiation) interact with and cause damage to
the genetic material of cells. Additionally, hints began to emerge that living
organisms can recover from the lethal effects of such damage 3. These advances
notwithstanding, “a combination of intellectual biases and to a lesser extent
political influences, constrained the emergence of DNA repair as an area of
investigative inquiry in parallel with other aspects of gene function. 2” For one
thing, the discovery that the master blueprint of life – the genetic material – was
made of DNA still lay a good 13 years ahead. “Genes were still presumed to be
made of proteins and to be intrinsically stable. There was no imperative to
consider them at special risk to environmental or spontaneous damage, and
hence in need of special biochemical perturbations. Mutations were considered
to be rare events that were of enormous pragmatic value for genetic studies, but
their mechanism of origin was not obviously experimentally tractable. Recovery
after exposure to X-rays and UV light was an anecdotal phenomenon at best,
and at worst the province of government “scientists” who were primarily intent
on gleaning useful biological applications for the militaristic use of radiation, a
task for which they were lavishly supported. Thus, the first direct experimental
evidence for DNA repair did not emerge until just before the middle of the 20th
century, and it was not until almost a decade later that the term DNA repair was
confidently and unambiguously incorporated into the lexicon of molecular and
cellular biology.
Importance
DNA in the living cell is subject to many chemical alterations (a fact often
forgotten in the excitement of being able to do DNA sequencing on dried and/or
frozen specimens. If the genetic information encoded in the DNA is to remain
uncorrupted, any chemical changes must be corrected.

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A failure to repair DNA produces a mutation.
The recent publication of the human genome has already revealed 130 genes
whose products participate in DNA repair. More will probably be identified
soon.
The causes and effects of DNA damage
The most significant consequence of oxidative stress in the body is thought to
be damage to DNA. DNA may be modified in a variety of ways, which can
ultimately lead to mutations and genomic instability. This could result in the
development of a variety of cancers including colon, breast, and prostate. Here
we discuss the various types of damage to DNA, including oxidative damage,
hydrolytic damage, DNA strand breaks, and others.
Oxidative DNA damage refers to the oxidation of specific bases. 8-
hydroxydeoxyguanosine (8-OHdG) is the most common marker for oxidative
DNA damage and can be measured in virtually any species. It is formed and
enhanced most often by chemical carcinogens. A similar oxidative damage can
occur in RNA with the formation of 8-OHG (8-hydroxyguanosine), which has
been implicated in various neurological disorders.
Hydrolytic DNA damage involves deamination or the total removal of
individual bases. Loss of DNA bases, known as AP (apurinic/apyrimidinic)
sites, can be particularly mutagenic and if left unrepaired they can inhibit
transcription. Hydrolytic damage may result from the biochemical reactions of
various metabolites as well as the overabundance of reactive oxygen species.
Ultraviolet and other types of radiation can damage DNA in the form of DNA
strand breaks. This involves a cut in one or both DNA strands; double-strand
breaks are especially dangerous and can be mutagenic, since they can
potentially affect the expression of multiple genes. UV-induced damage can
also result in the production of pyrimidine dimers, where covalent cross-links
occur in cytosine and thymine residues. The most common pyrimidine dimers
are cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone

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photoproducts (6-4PP). CPD and 6-4PP are the most frequent DNA mutations
found in the p53
proper protein in skin cancers. Pyrimidine dimers can disrupt polymerases and
prevent replication of DNA.
DNA damage may also result from exposure to polycyclic aromatic
hydrocarbons (PAHs). PAHs are potent, ubiquitous atmospheric pollutants
commonly associated with oil, coal, cigarette smoke, and automobile exhaust
fumes. A common marker for DNA damage due to PAHs is Benzo (a) pyrene
diol epoxide (BPDE). BPDE is found to be very reactive, and known to bind
covalently to proteins, lipids, and guanine residues of DNA to produce BPDE
adducts. If left unrepaired, BPDE-DNAadducts may lead to permanent
mutations resulting in cell transformation and ultimately tumor development.
The Comet Assay, or single cell gel electrophoresis assay (SCGE), is a common
technique used to measure all types of DNA damage, including the various
types of damage mentioned above. It is a convenient tool for measuring
universal DNA damage in individual cells.
Damage caused by exogenous agents comes in many forms. Some examples
are:
UV-B light causes crosslinking between adjacent cytosine and thymine bases
creating pyrimidine dimers. This is called direct DNA damage.
UV-A light creates mostly free radicals. The damage caused by free radicals is
called indirect DNA damage.
Direct Reversal Repair:-
Most cases of DNA damage are not reversible. For cases that are reversible, our
body uses direct reversal repair mechanism to correct the damaged base.
DNA Repair Mechanism

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Direct reversal repair is a mechanism of repair where the damaged area or lesion
is repaired directly by specialised proteins in our body. It is the simplest form of
DNA repair and also, the most energy efficient method. It does not require a
reference template unlike the other single-strand repair mechanism. Moreover,
it does not involve the process of breaking the phosphodiester backbone of the
DNA.
Direct repairing of O6-methylguanine.
An example of reversible DNA damage repairable via Direct Repair is
Alkylation which can be repaired via direct removal of the Alkyl groups.
Alkylating agents are carcinogens that is capable of alkylating DNA in our
body. It is widely used to create medicines (e.g., treatment of leukaemia, tumors
) and industrial chemicals.
Alkylated DNA bases resulted in improper base pairing and ultimately, lead to
cell death.
An example of Alkylation is Methylation which is the addition of a methyl
group (CH3) to a guanine (G) nucleotide. This resulted in a complementary
pairing to thymine (T) instead of cytosine (C).

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Direct repairing of Methylation.
Direct reversal repair involves using a sacrificial protein for the removal of each
Alkyl group since each protein is permanently inactivated upon transfer of alkyl
group to protein. An example of enzyme involved in direct reversal repair is
Methyltransferases (MGMT)
MGMT is a critical enzyme used in the direct reversal of DNA damage, O6-
alkylguanine in our body. It is a common protein found in all types of living
organisms ranging from prokaryotes to eukaryotes
1-Single strand damage:-
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.

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2-Base Excision Repair (BER)
The steps and some key players:
1. Removal of the damaged base (estimated to occur some 20,000 times a
day in each cell in our body) by a DNA glycosylase. We have at least 8 genes
encoding different DNA glycosylases each enzyme responsible for identifying
and removing a specific kind of base damage.
2. Removal of its deoxyribose phosphate in the backbone, producing a gap.
We have two genes encoding enzymes with this function.
3. Replacement with the correct nucleotide. This relies on DNA polymerase
beta, one of at least 11 DNA polymerases encoded by our genes.
4. Ligation of the break in the strand. Two enzymes are known that can do
this; both require ATP to provide the needed energy.
Nucleotide Excision Repair (NER)
NER differs from BER in several ways.
• It uses different enzymes.
• Even though there may be only a single "bad" base to correct, its
nucleotide is removed along with many other adjacent nucleotides; that is, NER
removes a large "patch" around the damage.
The steps and some key players:
1. The damage is recognized by one or more protein factors that assemble at
the location.
2. The DNA is unwound producing a "bubble". The enzyme system that
does this is Transcription Factor IIH, TFIIH, (which also functions in normal
transcription.
3. Cuts are made on both the 3' side and the 5' side of the damaged area so
the tract containing the damage can be removed.
4. A fresh burst of DNA synthesis using the intact (opposite) strand as a
template fills in the correct nucleotides. The DNA polymerases responsible are
designated polymerase delta and epsilon.

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5. A DNA ligase covalently inserts the fresh piece into the backbone.
3- Mismatch Repair (MMR)
Mismatch repair deals with correcting mismatches of the normal bases; that is,
failures to maintain normal Watson-Crick base pairing (A•T, C•G)
It can enlist the aid of enzymes involved in both base-excision repair (BER) and
nucleotide-excision repair (NER) as well as using enzymes specialized for this
function.
• Recognition of a mismatch requires several different proteins including
one encoded by MSH2.
• Cutting the mismatch out also requires several proteins, including one
encoded by MLH1.
Mutations in either of these genes predisposes the person to an inherited form of
colon cancer. So these genes qualify as tumor suppressor genes.
Cells also use the MMR system to enhance the fidelity of recombination; i.e.,
assure that only homologous regions of two DNA molecules pair up to cross
over and recombine segments (e.g., in meiosis)
4- Double-Strand Break Repair in Eukaryotes
Double-strand breaks in eukaryotes are probably the most dangerous form of
DNA damage. They are really broken chromosomes, and if they are not
repaired, they can lead to cell death or, in vertebrates, to cancer. Eukaryotic
cells deal with double-strand breaks in DNA (DSBs) in two ways: First, they
can use homologous recombination, with the unbroken sister chromatid as the
recombining partner. This mechanism is similar to recombination repair in
bacteria except that both strands must participate in recombination. Second,
eukaryotic cells can use non homologous end-joining (NHEJ). In replicating
cells in S and G2 phases, homologous recombination is the dominant

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mechanism, because only one DNA copy is broken and the other is available to
align the breaks properly.
Yeast cells, which divide frequently, rely primarily on homologous
recombination to repair their double-strand breaks. On the other hand,
mammalian cells in G1 phase preferentially use nonhomologous end-joining
because the DNA has not replicated and no second, homologous chromosome is
yet available to serve as a template for repair. In this section, we will focus on
the latter mechanism.
5-Non homologous end-joining
J. Phillips and W. Morgan investigated nonhomologous end-joining in 1994
by introducing a restriction endonuclease into Chinese hamster ovary cells. This
enzyme made double-stranded cuts in chromosomes, including a site within the
adenine phosphoribosyltransferase (APRT) gene, which was present in only one
copy in these cells. Then these workers looked for viable cells with mutations in
the APRT gene and sequenced the mutated genes to see what had happened
during the rejoining process. They found mostly short insertions and deletions
of DNA around the cleavage site. Furthermore, these insertions and deletions
appeared to have been directed by microhomology—small areas of homology
(1–6 bp)—in the DNA ends. Figure 20.34 shows a model for nonhomologous
end-joining that explains these and other fi ndings. First, the DNA ends attract
Ku, a dimer of two polypeptides (Ku70 [Mr 5 69 kD] and Ku80 [Mr 5 83 kD]).
One of the important functions of this protein is to protect the DNA ends from
Degradation until end-joining is complete. Ku has DNA-dependent ATPase
activity and is the regulatory subunit for DNA protein kinase (DNA-PK), whose
catalytic subunit is known as DNA-PKcs. X-ray crystallography studies have
shown that Ku binds to DNA ends like a ring on a finger. Its two subunits form
a ring that is lined with basic amino acids, which help it bind to acidic DNA.
Once Ku has bound to a DNA end, it can recruit the DNA-PKcs and perhaps
other proteins, completing the DNA-PK complex. The protein complexes on
each DNA end have binding sites, not only for the DNA ends, but also for
double-stranded DNA adjacent to the ends. Thus, these DNA-PK complexes, by
binding to the other DNA ends in the process that require ku.

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Coping with DNA Damage without Repairing It
The direct reversal and excision repair mechanisms described so far are all true
repair processes. They eliminate the defective DNA entirely. However, cells
have other means of coping with damage that do not remove it but simply skirt
around it. These are sometimes called repair mechanisms, even though they
really are not. A better term might be damage bypass mechanism. These
mechanisms come into play when a cell has not performed true repair of a
lesion, but has either replicated its DNA or both replicated its DNA and divided
before repairing the lesion. At each of these steps (DNA replication and cell
division), the cell loses attractive options for dealing with DNA damage and is
increasingly faced with more dangerous options.
Diagram of Non homologous end joining

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6- Recombination Repair
Recombination repair is the most important of these mechanisms. It is also
sometimes called post replication repair because replication past a pyrimidine
dimer can leave a problem: a gap opposite the dimer that must be repaired.
Excision repair will not work any longer because there is no undamaged DNA
opposite the dimer—only a gap—so recombination repair is one of the few
alternatives left. Diagram shows how recombination repair works. First, the
DNA is replicated. This creates a problem for DNA with pyrimidine dimers
because the dimers stop the replication machinery. Nevertheless, after a pause,
replication continues, leaving a gap (a daughter strand gap) across from the
dimer. (A new primer is presumably required to restart DNA synthesis.) Next,
recombination occurs between the gapped strand and its homolog on the other
daughter DNA duplex. This recombination depends on the recA gene product,
which exchanges the homologous DNA strands. We have encountered recA
before in our discussion of the induction of an l prophage during the SOS
response
The net effect of this recombination is to fill in the gap across from the
pyrimidine dimer and to create a new gap in the other DNA duplex. However,
because the other duplex has no dimer, the gap can easily be filled in by DNA
polymerase and ligase. Note that the DNA damage still exists, but the cell has at
least managed to replicate its DNA. Sooner or later, true DNA repair could
presumably occur.

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Global response to DNA damage
Cells exposed to ionizing radiation, ultraviolet light or chemicals are prone to
acquire multiple sites of bulky DNA lesions and double-strand breaks.
Moreover, DNA damaging agents can damage other biomolecules such as
proteins, carbohydrates, lipids, and RNA. The accumulation of damage, to be
specific, double-strand breaks or adducts stalling the replication forks, are
among known stimulation signals for a global response to DNA damage.The
global response to damage is an act directed toward the cells' own preservation
and triggers multiple pathways of macromolecular repair, lesion bypass,
tolerance, or apoptosis. The common features of global response are induction
of multiple genes, cell cycle arrest, and inhibition of cell division.
DNA damage checkpoint
A DNA damage checkpoint is a pause in the cell cycle that is induced in
response to DNA damage to ensure that the damage is repaired before cell
division resumes. Proteins that accumulate at the damage site typically activate
the checkpoint and halt cell growth at the G1/S or G2/M boundaries
SOS response in prokaryotes
The SOS response is a state of high-activity DNA repair, and is activated by
bacteria that have been exposed to heavy doses of DNA-damaging agents. Their
DNA is basically chopped to shreds, and the bacteria attempts to repair its
genome at any cost (including inclusion of mutations due to error-prone nature
of repair mechanisms). The SOS system is a regulon; that is, it controls
expression of several genes distributed throughout the genome simultaneously.
The primary control for the SOS regulon is the gene product of lexA, which
serves as a repressor for recA, lexA (which means it regulates its own
expression), and about 16 other proteins that make up the SOS response.
During a normal cell’s life, the SOS system is turned off, because lexA
represses expression of all the critical proteins. However, when DNA damage
occurs, RecA binds to single-stranded DNA (single-stranded when a lesion
creates a gap in daughter DNA). As DNA damage accumulates, more RecA
will be bound to the DNA to repair the damage.

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What is interesting is that RecA, in addition to its abilities in recombination
repair, stimulates the autoproteolysis of lexA’s gene product to do a bit That is,
LexA will cleave itself in the presence of bound RecA, which causes cellular
levels of LexA to drop, which, in turn, causes coordinate derepression
(induction) of the SOS regulon genes.
As damage is repaired, RecA releases DNA; in this unbound form, it no longer
causes the autoproteolysis of LexA, and so the cellular levels of LexA rise to
normal again, shutting down expression of the SOS regulon genes.
Eukaryotic transcriptional responses to DNA damage
Eukaryotic cells exposed to DNA damaging agents also activate important
defensive pathways by inducing multiple proteins involved in DNA repair, cell
cycle checkpoint control, protein trafficking and degradation. Such genome
wide transcriptional response is very complex and tightly regulated, thus
allowing coordinated global response to damage. Exposure of yeast
Saccharomyces cerevisiae to DNA damaging agents results in overlapping but
distinct transcriptional profiles. Similarities to environmental shock response
indicates that a general global stress response pathway exist at the level of
transcriptional activation. In contrast, different human cell types respond to
damage differently indicating an absence of a common global response. The
probable explanation for this difference between yeast and human cells may be
in the heterogeneity of mammalian cells. In an animal different types of cells
are distributed among different organs that have evolved different sensitivities
to DNA damage.
Hereditary DNA repair disorders
Defects in the NER mechanism are responsible for several genetic disorders,
including:
Xerodema 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.

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Mental retardation often accompanies the latter two disorders, suggesting
increased vulnerability of developmental neurons.
Other DNA repair disorders include:
•Werner's syndrome: premature aging and retarded growth
•Bloom's syndrome: sunlight hypersensitivity, high incidence of malignancies
(especially leukemias).
•Ataxia telangiectasia: sensitivity to ionizing radiation and some chemical
agents
All of the above diseases are often called "segmental 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
anaemia, hereditary breast cancer and hereditary colon cancer.
Repair of damaged DNA
When the new strand containing the mismatch is identified, an endonuclease
nicks the mismatched strand, and the mismatched base(s) is/are removed. The
gap left by removal of the mismatched nucleotide(s) is filled, using the sister
strand as a template, by a DNA polymerase (DNA polymerase in E. coli)- The
3'-hydroxyl of the newly synthesized DNA is spliced to the 5'-phosphate of the
remaining stretch of the original DNA strand by DNA ligase (see p. 403).
[Note: A defect in mismatch repair in humans has been shown to cause
hereditary nonpolyposis colon cancer (HNPCC), one of the most common
inherited cancers.
Repair of damage caused by ultraviolet light
Exposure of a cell to ultraviolet light can result in the covalent joining of two
adjacent (usually thymines), producing a These thymine prevent DNA

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polymerase from replicating the DNA strand beyond the site of dimer
formation. Thymine dimers are excised in bacteria as illustrated. A similar
pathway is present in humans.
1. Recognition and excision of dimers by UV-specific endonuclease: First, a
UV-specific endonuclease (called uvrABC ase) recognizes the dimer, and
cleaves the damaged strand at phosphodiester bonds on both the 5'-side and 3'-
side of the dimer. The damaged oligonucleotide is released, leaving a gap in the
DNA strand that formerly contained the dimer. This gap is filled in using the
same repair system described above.
2. Ultraviolet radiation and cancer: Pyrimidine dimers can be formed in the skin
cells of humans exposed to unfiltered sunlight. The rare genetic disease
xeroderma pigmentosum, the cells cannot repair the damaged DNA, resulting in
extensive accumulate- the rare genetic disorder.
Exposure of a cell to ultraviolet light can result in the covalent joining of two
adjacent (usually thymine’s), producing a These thymine prevent DNA
polymerase from replicating the DNA strand beyond the site of dimer
formation. A similar pathway is present in humans
Correction of base alterations (base excision repair)
The bases of DNA can be altered, either spontaneously, as is the case with
cytosine, which slowly undergoes deamination (the loss of its amino group) to
form uracil, or by the action of or alkylation compounds. For example, nitrous
acid, which is formed by the cell from precursors, such as the nitrites, and
nitrates, is a potent compound that cytosine, adenine, and guanine. Bases can
also be lost spontaneously. For example, approximately 10,000 purine bases are
lost this way per cell per day. Lesions involving base alterations or loss can be
corrected by the following mechanisms.
1. Removal of abnormal bases: Abnormal bases, such as uracil, which can
occur in DNA either by deamination of cytosine or improper
incorporation of instead
2. of dTTP during DNA syn- thesis, are recognized by specific that
hydrolytically cleave them from the deoxyribose-phosphate backbone of

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the strand. This leaves an apyrimidinic site (or apurinic, if a purine cleave
them from the deoxyribose-phosphatebackbone of the strand. This leaves
an apyrimidinic site (or apurinic, if a purine was removed), referred to as
an AP-site.
2. Recognition and repair of an AP-site: Specific recognize that a base is
missing and initiate the process of exci-sion and gap filling by making an
endonucleolytic cut just to the 5' side of the AP-site. A deoxyribose-phosphate
lyase removesthe
single, empty, sugar-phosphate residue. DNA polymerase in E. and DNA ligase
complete the repair process.
Repair of double-strand breaks
High-energy radiation or oxidative free radicals (see p. 145) can cause double-
strand breaks in DNA, which are potentially lethal to the cell. Double-strand
breaks also occur naturally during gene rear- arrangements. Double-strand DNA
breaks cannot be corrected by the previously described strategy of excising the
damage on one strand and using the remaining strand as a template for replacing
the miss- ing nucleotide(s). Instead, double-strand breaks are repaired by one of
two systems. The first is nonhomologous end-joining repair, in which the ends
of two DNA fragments are brought together by a group of proteins that effect
their religation. This system does not require that the two DNA sequences have
any sequence homology. However, this mechanism of repair, which is the main
repair mecha-nism in humans, is error prone and mutagenic. Defects in this
repair system are associated with a predisposition to cancer and immune
deficiency syndromes. The second repair system, homologous recombination
repair, uses the enzymes that normally perform genetic recombination between
homologous chromosomes during this system is used predominantly by the
lower eukaryotes to repair double-strand breaks.