General classes of DNA damage

mutagen - any chemical agent that

causes an increase in the rate of
mutation above the spontaneous
background
Spontaneous damage to DNA: can
occur through the action of water in
the aqueous environment of the cell.
mutagens have a major impact on
public health

Major classes of DNA damage:

1. single base changes
2. structural distortion
3. DNA backbone damage

Single base changes

single base change or conversion - affects the DNA
sequence, but has only a minor effect on overall structure
For example: deamination - replacement of the amino
group of cytosine with oxygen converts cytosine to uracil
Deamination - most frequent and important kind of
hydrolytic damage, and can occur spontaneously from the
action of water, or be induced by a chemical mutagen.
When a UG base pair replaces a CG base pair - causes
only a minor structural distortion in the DNA double helix.
type of damage that is not likely to completely block the
process of replication or transcription, but may lead to the
production of a mutant RNA or protein product.

Single base changes

5-methylcytosine in place of cytosine
found frequently in vertebrate DNA.
Methylated cytosines - hotspots for
spontaneous mutations in vertebrate
DNA because deamination of 5methylcytosine generates thymine.
change of a GC base pair into an AT
when damaged DNA is replicated.

Single Base Changes

Other processes that damage the DNA: alkylation,
oxidation, and radiation.
Nitrosamines example of alkylating agents that
lead to the formation of O6-methylguanine.
Methylguanine - mispairs with thymine, resulting
in the change of a GC base pair into an AT base
pair when the damaged DNA is replicated.

Single Base Changes

Potent oxidizing agents - generated by ionizing
radiation and by chemical agents that generate
free radicals.
reactive oxygen species (O2 -, H2O2, and OH[])
can generate 8-oxoguanine (oxoG)
Oxoguanine (OxoG) - a damaged guanine base
containing an extra oxygen atom that is highly
mutagenic because it can form a Hoogsteen base
pair with adenine. a GC TA transversion,
which is one of the most common mutations
found in human cancers.

Structural distortion
Ultraviolet (UV) light has a detrimental effect on cells due to
selective absorption of the UV rays.
Radiation with a wavelength of about 260 nm is strongly
absorbed by the bases.
most frequent UV-induced lesions of DNA: induction of
pyrimidine dimers between two neighboring thymine bases by
UV irradiation
These are also termed cyclobutanepyrimidine dimers (CPD),
because a cyclobutane ring is generated by links between
carbon atoms 5 and 6 of adjacent thymines.
Because covalent bonds form between thymines on the same
strand, this disrupts the complementary base pairs that form
the double helix.
Thymine dimers thus distort the structure of the duplex DNA.

 Effects of structural distortions:

impede transcription and replication by
blocking the movement of polymerases
induction of pyrimidine dimers is a more
severe defect than a single base change
pyrimidine (6,4)-pyrimidone photoproducts
- UV irradiation-induced dimers between
cytosine and thymine
Other bulky adducts can be induced by
chemical mutagenesis; e.g. by exposure to
large polycyclic hydrocarbons or alkylating
agents

 Other substances that cause DNA damage:

intercalating agents and base analogs.
Intercalating agents such as ethidium bromide
contain several flat polycyclic rings that insert
between the DNA bases, binding and stacking
with the DNA bases, just as the bases bind or
stack with each other in the double helix.
Due to the resulting distortion of the double helix,
intercalating agents can cause insertion or
deletion of one or more base pairs during DNA
replication.
Base analogs are compounds such as 5bromouracil, an analog of thymine, that
substitute for normal bases. They are similar
enough to the normal bases to be taken up by
cells, converted into deoxynucleotides, and
incorporated into DNA during replication.

DNA backbone damage

Effects of Backbone damage: formation of abasic sites (loss of the
nitrogenous base from a nucleotide) and double-strand DNA breaks.
Abasic sites - generated spontaneously by the formation of unstable
base
adducts.
For example, in purine nucleotides, the sugarpurine bonds are
relatively labile.
Hydrolysis of the N-glycosyl linkage in the purine base by the action of
water leaves a hydroxyl (-OH) in its place in the depurinated DNA.
Double-strand breaks can be induced by ionizing radiation (e.g. X-rays,
radioactive materials) and a wide range of chemical compounds.
Ionizing radiation can attack (ionize) the deoxyribose sugar in the DNA
backbone directly or indirectly by generating reactive oxygen species.
Double-strand breaks are the most severe type of DNA damage, since
they disrupt both DNA strands.

Cellular response to DNA

damage
Responses to different types of DNA
damage fall into three main categories:
1. those that bypass the damage
2. those that directly reverse the
damage
3. those that remove the damaged
section of DNA and replace it in with
undamaged DNA, by excision or
recombinational repair systems.

esion bypass

 The normal replication machinery uses high-fidelity DNA polymerases. These high-fidelity
polymerases accurately copy nondamaged template DNA, but are unable to bypass DNA
lesions that cause structural distortion of the DNA helix.
Specialized low-fidelity, error-prone DNA polymerases (see Table 6.1)
transiently replace the replicative polymerases and copy past damaged DNA in a process
called translesion
synthesis (TLS) (Fig. 7.4).
In the simplest of models, the replicative polymerase, being unable to bypass a
lesion in the DNA either falls off the DNA or simply translocates downstream of the lesion
to continue
replication.
his allows for proliferating cell nuclear antigen (PCNA) mediated loading of another DNA
polymerase capable of replicating the lesion. In keeping with the factory model for DNA
replication, TLS
polymerases and their auxiliary factors are stored in these subnuclear compartments for
rapid recruitment.
Eventually, the replicative polymerase regains control of the template until such time as
replication is
completed or the polymerase again encounters a replication-blocking DNA lesion.

 The error-prone DNA polymerases are able to copy damaged DNA templates permissively
and efficiently.
However, because they are error-prone they may generate mutations. Typical error rates
range from 10-1
to 10-3 per base pair (on undamaged DNA). Most lesions completely alter the pairing
properties of the
pre-existing bases. In this case, the polymerases insert incorrect nucleotides opposite the
lesions, giving rise
to nucleotide substitution mutants. Alternatively, when the polymerases skip past
lesions, they insert a correct
nucleotide opposite bases downstream from the lesion, generating a frameshift
mutation. An exception to
the general tendency of repair polymerases to be error-prone is DNA polymerase eta ().
DNA polymerase
performs TLS past a thyminethymine (TT) dimer by inserting two adenine residues.
This results in the
lesion being bypassed in an error-free manner. The presence of DNA polymerase thus
protects cells from
UV damage. In contrast, DNA polymerase iota () achieves TLS by a highly error-prone
bypass of a TT dimer.

 Translesion synthesis enables a cell to survive

what might be a fatal block to replication, but with
the
risk of a higher mutation rate. For this reason, in
E. coli, the translesion polymerases (DNA
polymerases IV
and V; see Focus box 6.1) are not present under
normal circumstances. Their synthesis is only
induced in
response to DNA damage, as part of a pathway
known as the SOS response.

Direct reversal of DNA damage

In most organisms, UV radiation damage to DNA (pyrimidine dimers) can
be directly repaired by a process
called photoreactivation or light repair. However, placental mammals,
including humans, do not have a
photoreactivation pathway. During photoreactivation, the enzyme DNA
photolyase uses energy from nearUV to blue light to break the covalent
bonds holding the two adjacent pyrimidines together (Fig. 7.5).
Another example of direct reversal of DNA damage is the repair of the
methylated base O6methylguanine. Methyltransferases present in organisms ranging from E.
coli to humans catalyze removal of
the methyl group (Fig. 7.6). A sulfhydryl group of a cysteine residue of the
methyltransferase accepts the
methyl group from guanine. This process is very costly to the cell
because once the methyltransferase accepts
the methyl group from guanine, the enzyme cannot be used again.

Repair of single base changes and structural

distortions
by removal of DNA damage
Repair systems that remove damaged DNA include repair
of single base changes, structural distortions, and
double-stranded DNA damage (see Table 7.2). A key
feature of each of these pathways is that multiple
dynamic protein interactions are involved in the repair
process. There is an ordered hand-off of DNA from
one protein or complex to another. The DNA repair proteins
are modular, composed of multiple structural
domains with distinct biochemical functions. They also
have multiple binding sites of modest affinity that
mediate interaction with other repair proteins. The
presence of multiple binding sites facilitates trading
places by direct competition for binding sites or allosteric
structural rearrangements.