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Mutations and DNA damage
Mutations and DNA damage
Mutations result from
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Changes in the nucleotide sequence of DNA
Deletions
Insertions
Rearrangements of DNA sequences in the genome.
These changes can be:
• Spontaneous mutations
that occurs as a result of natural processes in cells, for
example DNA replication errors
• Induced mutations
Occur as a result of interaction of DNA with an outside agent
that causes DNA damage (UV radiation, oxygen free radical, chemicals).
Spontaneous mutations
Point mutations
The simplest type of mutation in which a nucleotide pair in a DNA duplex is replaced
with a different nucleotide pair.
Whether nucleotide substitutions have a phenotypic effect depends on where they are
located in the DNA sequence.
Transition mutations replace one pyrimidine base with another, or one purine base with
another.
Transversion mutations replace a pyrimidine with a purine or vice versa.
Both mutations can be permanently incorporated by DNA replication.
Point mutations:
Silent and nonsense mutations
Mutations that change the nucleotide sequence without changing
the amino acid sequence are called synonymous mutations or silent
mutations.
Mutational changes in nucleotides that are outside of
coding regions can also be silent.
However, some noncoding sequences do have essential
functions in gene regulation and, in this case, mutations in
these sequences would have phenotypic effects.
A nucleotide substitution that creates a new stop codon is called a
nonsense mutation.
It causes premature chain termination during protein synthesis.
Nearly always a nonfunctional product.
Point mutations: Missense mutations
Nucleotide substitutions in protein-coding regions that result in changed
amino acids are called nonsynonymous mutations or missense mutations.
May alter the biological properties of the protein.
The human hereditary disease sickle cell anemia is an A→T transversion which
changes the codon for glutamic acid to valine in the -globin chain of the
hemoglobin.
It results in a decrease in the ability of red blood cells to carry oxygen
throughout the body.
Most people have only hemoglobin type A (Hb A) within RBC (normal genotype:
Hb AA). In Sickle-cell anemia there is homozygosity for the mutation that
causes HbSS.
Point mutations:
Insertions or deletions can cause frameshift
mutations
If the length of an insertion or deletion is not an exact multiple of three nucleotides,
this results in a shift in the reading frame of the resulting mRNA.
Usually leads to production of a nonfunctional protein.
Example: human hereditary disease cystic fibrosis.
The protein encoded by CFTR gene (cystic fibrosis
transmembrane conductance regulator) is required to
regulate the components of sweat, digestive fluids, and
mucus. CFTR regulates the movement of chloride and
sodium ions across epithelial membranes, such as the
alveolar epithelia located in the lungs.
The deletion of three base pairs in the nucleotide
sequence of CFTR gene results in the loss of the
codon for phenylalanine.
CF develops when neither gene works normally (as a result of mutation) and
therefore has autosomal recessive inheritance
Induced mutations
General classes of DNA damage
Spontaneous damage to DNA can occur through the action of
water in the aqueous environment of the cell. Degradation of
DNA in water is due to DNA hydrolysis, that is, the breaking
of DNA bonds through the addition of water. This type of
damage occurs very rarely.
A mutagen is any chemical agent that causes an increase in
the rate of mutation above the spontaneous background.
Three general classes of DNA damage
• Single base changes
• Structural distortion
• DNA backbone damage
Induced mutations
Single base changes
Single base changes: deamination
A single base change or “conversion” affects the DNA sequence
but has only a minor effect on overall structure.
Deamination is the most frequent and important kind of hydrolytic
damage, and can occur spontaneously from the action of water, or
be induced by a chemical mutagen. The replacement of the amino
group of cytosine with oxygen converts cytosine to uracil.
When a UG base pair replaces a CG
base pair this causes only a minor
structural distortion in the DNA
double helix.
This type of damage 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: deamination
Vertebrate DNA frequently contains 5-methylcytosine in place of
cytosine.
Methylated cytosines are “hotspots” for spontaneous mutation in
vertebrate DNA because deamination of 5-methylcytosine
generates thymine.
This results in the change of a GC base pair into an AT when
damaged DNA is replicated
Single base changes: alkylation
Alkylation is the transfer of an alkyl group from one molecule
to another.
Alkyl groups range from single carbon compounds such as methyl
groups to much longer chains of hydrocarbons.
Alkylating agents such as nitrosamines lead to the formation of
O6-methylguanosine.
This modified base often mispairs with thymine.
Can result in a GC→GT→AT point mutation after DNA replication.
Single base changes: oxidation
Oxidation is any chemical reaction in
which a compound loses electrons.
Potent oxidizing agents are generated by
ionizing radiation and by chemical agents
that generate free radicals.
These reactive oxygen species (O2−,
H2O2, and OH[·]) can generate 8oxoguanine (oxoG), a damaged guanine
base containing an extra oxygen atom.
OxoG can form a Hoogsteen base pair
with adenine.
Gives rise to a GC→TA transversion.
One of the most common mutations found
in human cancers.
Induced mutations
Structural distortion
Structural distortion
Thymine dimer
UV radiation induces the
formation of a cyclobutane ring
between adjacent thymines,
forming a T-T dimer.
The T-T dimer distorts the
double helix and can block
transcription and replication.
UV radiation can also induce
dimers between cytosine and
thymine.
Structural distortion
Structural distortion can be caused by intercalating agents and
base analogs:
Ethidium bromide has several flat
polycyclic rings that insert between
the DNA bases.
5-bromouracil, an analog of thymine,
can mispair with guanine.
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.
Induced mutations
DNA backbone damage
DNA backbone damage
Formation of abasic sites
Loss of the nitrogenous base from a nucleotide.
Generated spontaneously by the formation of unstable base
adducts.
Double-stranded DNA breaks
Induced by ionizing radiation (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 responses to DNA damage
Damage bypass
Damage reversal
Damage removal
Cellular responses to DNA damage
Damage bypass
Lesion 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
transiently replace the replicative polymerases and copy past
damaged DNA in a process called Translesion synthesis (TLS).
Lesion bypass
(1) The replication
machinery is shown arrested
(stop sign) at a thymine
dimer on the template
for leading strand synthesis.
Multiple specialized errorprone DNA repair
polymerases are stored in a
subnuclear
compartment near the
arrested replication fork
(DNA pol ι, λ, ζ, η, and κ).
(2) When the replication
fork stalls at the lesion,
DNA pol δ is replaced by
one of the specialized
polymerases (DNA pol ι in
this example).
Lesion bypass
(3) When translesion
synthesis has bypassed the
damage and extended to a
suitable position downstream,
another polymerase switch
occurs.
DNA pol ι is released and
replaced by DNA pol δ.
High-fidelity DNA replication
continues
Lesion bypass
Error-prone DNA polymerases
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.
Most lesions completely alter the pairing properties of
the
pre-existing bases.
In this case, the polymerases
May insert incorrect nucleotides opposite the lesion:
nucleotide substitution
May skip past and insert correct nucleotides opposite
bases downstream: frameshift
Lesion bypass
An exception to the general tendency of repair polymerases
to be error-prone is DNA polymerase eta (η)
DNA polymerase eta ()
• Performs translesion synthesis past TT dimers by
inserting AA. This results in the lesion being bypassed in
an error-free manner
• Has an extra wide active site that can accommodate two
dNTPs instead of one.
• Van der Waals forces and hydrogen-bonding interactions
hold the TT dimer so that the two thymines can be paired
with two adenines.
• The T-T dimer is still present in the original parent
strand of the DNA double helix after translesion
synthesis
Lesion bypass
Translesion synthesis enables a cell to survive what might
be a fatal block to replication, but with the risk of a
higher mutation rate.
Lesion bypass is not really a repair pathways; for example, the
T-T dimer is still present in the original parent strand of the
DNA double helix after translesion synthesis.
What pathways are available to repair such damage????
Cellular responses to DNA damage
Damage reversal
Reversal of thymine-thymine dimers by DNA
photolyase
In most organisms, UV radiation damage
to DNA can be directly repaired by a
process called photoreactivation or
“light repair”.
DNA photolyase uses energy from near
UV to blue light to break the covalent
bonds holding two adjacent pyrimidines
together.
How does this take place?????
Reversal of thymine-thymine dimers by DNA
photolyase
DNA photolyase has two cofactors:
• A pigment that absorbs UV/blue light
• Fully reduced flavin dinucleotide (FADH-)
The TT dimer is flipped out of the DNA helix and brought very
close to FADH-.
An electron is transferred from FADH- and the dimer is split.
Placental mammals, including humans, do not have a
photoreactivation pathway!!!!
Damage reversal by DNA methyltransferase
Methyltransferase catalyzes the transfer of the methyl group on O6methylguanine to the sulfhydryl group of a cysteine residue on the enzyme.
Methyltransferase are present in all organism
DNA methyltransferase binds
the minor groove of the DNA.
The O6-methylguanine flips out
from the double helix into the
active site.
A sulfhydryl group of a cysteine
in the active site accepts the
methyl group from guanine.
Once the methyltransferase accepts the methyl group from guanine,
the enzyme cannot be used again.
Cellular responses to DNA damage
Damage removal
Repair systems that remove damaged DNA include:
Repair of single base changes by two main pathways:
• Base excision repair
Involves the correction of single base changes that are
due to conversion.
• Mismatch repair
Mismatched base pairs that result from DNA polymerase
errors during replication are corrected by mismatch repair
Repair of structural distortion
• Nucleotide excision repair
The nucleotide excision repair pathway is used for repair of
structural distortion, for example bulges from thymine
dimers induced by UV irradiation
Repair of double-strand breaks
• Repaired by homologous recombination or nonhomologous endjoining.
Repair of single base changes
Base excision repair
Base excision repair
Base excision repair is initiated by a group of enzymes called DNA
glycosylases.
These enzymes cleave the N-glycosidic bond connecting the
deoxyribose sugar to the damaged base.
There are DNA glycosylases that recognize oxidized/reduced
bases, methylated bases, deaminated bases, or base mismatches.
For example, uracil DNA glycosylase removes uracil from UA or UG
base pairs, and the human oxoG repair enzyme, hOGG1, catalyzes
excision of oxoG.
The first step in base excision repair is recognition of the lesion
How do DNA repair proteins find the rare sites
of damage in a vast expanse of normal DNA is
poorly understood overall.
Base excision repair
Model for DNA damage recognition by 8-oxoguanine DNA glycosylase 1
(hOGG1)
DNA glycosylase 1 first binds nonspecifically to DNA. The damaged
base goes through a series of “gates” or checkpoints within the enzyme.
If the enzyme encounters a normal GC base pair, then:
The G is transiently extruded into a G-specific pocket and returned to
the double helix.
If the enzyme encounters a oxoG-C base pair, then:
The oxoG is extruded into the G-specific pocket and then inserted into
a lesion recognition pocket where it is excised.
Base excision repair
The excision of the damaged base form an abasic site in the DNA
Repair of deamination of cytosine to uracil
The next step in repair is to remove the abasic nucleotide.
In mammalian cells, the repair patch may be a single nucleotide
(short patch) or 2–10 nucleotides in lenght (long patch).
Base excision repair
Short patch repair
The enzymes
glycosylase-associated β-lyase and
APE1 (apurinic/apyrimidinic
endonuclease)
make nicks 3′ and 5′ to the abasic
site in the DNA , respectively.
DNA polymerase β replaces the
missing nucleotide, and the
DNA ligase 3–XRCC1 complex
seals the gaps in the sugar–
phosphate backbone.
Base excision repair
Long patch repair
APE1 makes an incision 5′ to the abasic
site.
Then, DNA polymerase δ or ε, and PCNA,
displace the strand 3′ to the nick to
produce a flap of 2–10 nt.
The flap is cut at the junction of the
single to double strand transition by FEN1 (flap endonuclease-1).
A patch of the same size is then
synthesized by DNA polymerase δ or ε
with the aid of PCNA and ligated by DNA
ligase I.
Repair of single base changes
Mismatch repair
Mismatch repair
Mismatch repair corrects mistakes that occur during DNA replication
and are not proofread by the DNA polymerase.
Basic features of the mismatch repair pathway are conserved from E.
coli to humans.
Mismatch repair pathway in mammalian cells
The first step in mismatch repair is the recognition of error by two
proteins the MutS/MutL complex.
Once the mismatch is recognized, a large region of DNA including the
mismatch is excised.
Mismatch repair
One model proposes that MutS /MutL then diffuses for several
thousand nucleotides either 5′ or 3′.
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A 5′ or 3′ single-strand break is generated by EXO1 in association
with PCNA (the replication clamp) and RFC (the clamp loader).
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5′→3′ or 3′→5′ progessive exonuclease activity of EXO1 removes
the mismatch.
Mismatch repair
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5′→3′ repair synthesis is mediated by DNA polymerase and
associated factors.
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Ligation of the remaining gap is catalyzed by DNA ligase I.
Reconstitution of mismatch repair in an in vitro system showed
that the recognition protein MutS, the exonuclease EXO1 and
DNA polymerase  are indispensible for repair.
Repair of structural distortion
Nucleotide excision repair
Nucleotide excision repair
Repair of structural distortion (e.g. bulges from thymine-thymine
dimers induced by UV irradiation).
Basic features of the nucleotide excision repair pathway are
conserved from E. coli to humans.
Xeroderma pigmentosum
• Autosomal recessive disorder.
• Photosensitivity.
• Greatly increased risk of sunlightinduced skin cancer.
• Neurological degeneration.
• Defects in nucleotide excision repair
or in T-T dimer translesion
synthesis.
Mammalian nucleotide excision repair pathway
The repair pathway responsible for recognizing lesions in the whole genome is
called global genome repair (GGR), while the transcription-coupled repair (TCR)
pathway identifies lesions in the transcribed strand of active genes.
In the first step, DNA damage is recognized by the cooperative binding of
XPC, RPA, XPA, and TFIIH.
• XPC binds first, followed by the other proteins (XPC, sliding along DNA
molecule and jumping from one DNA molecule to another, is able to scan the
genome rapidly and efficently) .
• TFIIH is a multiprotein complex that also plays an important role in gene
transcription.
Mammalian nucleotide excision repair pathway
Unwinding of the duplex DNA is promoted by the action of XPB
and XPD helicases, which are subunits of the TFIIH complex.
The endonuclease XPG
makes a 3′ incision
The endonuclease XPFERCC1 makes another 5′
incision approximately 20
nucleotides from the
damage.
Mammalian nucleotide excision repair pathway
After the helicase activity of XPB and XPD unwinds the duplex
DNA, the damage-containing DNA sequence (24–32 nt) is released.
Repair synthesis is mediated by DNA polymerase  or .
Ligation of the remaining gap in the DNA backbone is mediated by
DNA ligase I.
Repair of double-strand breaks
Homologous recombination
Double-strand break repair by removal of DNA
damage
This type of DNA damage is the most harmful to cells and
is often linked to cell death or cancer! For example,
hereditary deficiencies in double-strand break repair are
linked to an increased susceptibility to breast cancer.
Double-strand breaks in DNA are induced by reactive oxygen
species, ionizing radiation, and chemicals the generate reactive
oxygen species (free radicals).
Double-strand breaks in DNA are repaired by:
Homologous recombination
Repairs double-strand breaks by retrieving genetic information
from an undamaged homologous chromosome.
Nonhomologous end-joining (NHEJ)
Rejoins double-strand breaks via direct ligation of the DNA
ends without any requirement for sequence homology.
Double-strand break repair by removal of DNA
damage
• Homologous recombination plays a major role in doublestrand break repair in prokaryotes and single-cell
eukaryotes.
• In mammalian cells, double-strand breaks are
primarily repaired through NHEJ.
• In mammalian cells, the main function of homologous
recombination is to repair double-strand breaks at
the replication fork
Homologous recombination:
repair of double-strand breaks in DNA
Exposure of cells to ionizing radiation or other double strand
break-inducing agents triggers an increase in ATM kinase
activity (a serine–threonine kinase in the nucleus).
A kinase is a type of enzyme that transfers phosphate groups
from high-energy donor molecules, such as ATP, to specific
substrates, a process referred to as phosphorylation.
ATM is recruited to the break site and phosphorylates some
of the proteins involved in DNA repair and cell cycle control.
Humans that lack ATM suffer from a syndrome called ataxia
telangiectasia, characterized by extreme sensitivity to
radiation, increased susceptibility to developing cancer,
immunodeficiency, premature aging, and neurodegenerative
disorders.
Model for mammalian DNA double-strand break
repair by homologous recombination
A double-strand break (DSB) is induced by ionizing radiation.
The MRN (Mre11–Rad50–Nbs1) complex is rapidly recruited to
the DSB site.
Model for mammalian DNA double-strand break
repair by homologous recombination
MRN complexes form a bridge between
free DNA ends via the coiled-coil arms
of Rad50 dimers.
Inactive ATM dimers are recruited to
the DSBs through interaction with the
carboxyl terminus of Nbs1, and by a less
stable interaction with Rad50.
Activating signals are delivered to ATM
dimers, possibly through a
conformational change in Nbs1.
Model for mammalian DNA double-strand break
repair by homologous recombination
ATM undergoes phosphorylation
accompanied by its conversion from a
dimer to a monomer.
Activated ATM monomers either remain
near the DSB, where they phosphorylate
proteins involved in DNA repair, or
diffuse away from the DSB sites to
phosphorylate nuclear substrates, such
as p53 and Creb that are involved in cell
cycle control.
Model for mammalian DNA double-strand break
repair by homologous recombination
The 3′,5′-exonuclease activity of Mre11 generates 3′
single-strand DNA tails that are recognized by Rad52.
Strand invasion of the 3′ tails with intact homologous sequences is
initiated by Rad51.
The proteins Rad54, Rad55, Rad57, BRCA1, and BRCA2 are also involved
in homologous recombination, but their precise roles have yet to be
determined.
Model for mammalian DNA double-strand break
repair by homologous recombination
Strand exchange generates a hybrid molecule between damaged and undamaged
duplex DNAs.
Sequence information that is missing at the DSB site is restored by DNA
synthesis (newly synthesized DNA is shown in red). The interlinked molecules are
then processed by branch migration.
Model for mammalian DNA double-strand break
repair by homologous recombination
Heteroduplex DNA: duplex DNA formed during recombination is composed
of single DNA strands originally derived from different homologs.
Holliday junction: an intermediate in which the two recombining duplexes
are joined covalently by single crossovers.
The Holliday junction is resolved into two duplexes by an
enzyme complex called the resolvasome.
The resolvasome has “resolvase” activity.
Repair of double-strand breaks
Nonhomologous end-joining
Nonhomologous end-joining
In mammals double-strand breaks in DNA are primarily
repaired through nonhomologous end-joining.
This is thought to be the major pathway for repair of
double-strand breaks induced by ionizing radiation.
Following a double-strand break, the broken ends of DNA
are recognized by two heterodimers of the Ku70 and Ku80
proteins.
The heterodimers form a scaffold that holds the broken
ends in close proximity, allowing other enzymes to act.
Nonhomologous end-joining
The Ku heterodimer recruits the nuclease (Artemis/DNA-PKCS),
the polymerases
(DNA polymerases μ and λ), and the ligase complex (XRCC4–
DNA ligase IV) to the damaged site. The
endonuclease Artemis is activated after it is phosphorylated by
the DNA-dependent protein kinase catalytic
subunit (DNA-PKCS)
Nonhomologous end-joining
The activated Artemis/DNA-PKCS complex then trims excess or
damaged DNA at the break site. DNA polymerases μ and λ, or the
enzyme TdT are required for any nonhomologous end-joining events
that need fill-in of gaps or extension of the 3′ or 5′ overhangs.
The rejoining of the broken ends is carried out by DNA ligase IV in
association with XRCC4.
Nonhomologous end-joining
• This double-strand break repair process can lead to
mutation.
• Two broken ends can be ligated together regardless of
whether they came from the same chromosome.
• Frequently results in insertions or deletions at the break
site.
• Trade-off between repair and otherwise lethal breaks in
the genome.