DNA damage,
cellular sensing/responding,
and repair
Rebecca Fry, Ph.D.
DNA damage
DNA damage, due to
environmental factors
and normal metabolic
processes inside the
cell, occurs at a rate
of 1,000 to 1,000,000
molecular lesions per
cell per day.
DNA Damage
While this constitutes only 0.000165%
of the human genome's approximately 6
billion bases (3 billion base pairs)…
unrepaired lesions in critical genes (such
as tumor suppressor genes) can impede a
cell's ability to carry out its function
and appreciably increase the likelihood
of tumor formation.
Failure to repair DNA lesions may result
in blockages of transcription and
replication, mutagenesis, and/or
cellular cytotoxicity.
In humans, DNA damage has been shown
to be involved in a variety of genetically
inherited disorders, in aging, and in
carcinogenesis.
Sources of DNA Damage
DNA damage can be subdivided into two
main types:
ENDOGENOUS and EXOGENOUS
Endogenous sources of DNA Damage
endogenous damage such as attack by reactive
oxygen species produced from normal
metabolic byproducts
Types of Damage
The main types of damage to DNA due to endogenous
cellular processes:
1. oxidation of bases [e.g. 8-oxo-7,8-dihydroguanine (8oxoG)] In living cells ROS are formed continuously as a
consequence of metabolic and other biochemical
reactions . These ROS include superoxide (O2–·),
hydrogen peroxide (H2O2), hydroxyl radicals (OH·) and
singlet oxygen (1O2)
2. alkylation of bases (usually methylation), such as
formation of 7-methylguanine, 1-methyladenine, O6
methylguanine
3. hydrolysis of bases, such as deamination, depurination
and depyrimidination.
4. "bulky adduct formation" (i.e. benzo[a]pyrene
diol epoxide-dG adduct).
5. mismatch of bases, due to errors in DNA
replication, in which the wrong DNA base is
stitched into place in a newly forming DNA
strand, or a DNA base is skipped over or
mistakenly inserted.
Exogenous sources of DNA damage
caused by external agents such as
• ultraviolet [UV 200-300nm] radiation from the sun
• other radiation frequencies, including x-rays and
gamma rays
• human-made mutagenic chemicals
• cancer chemotherapy and radiotherapy
What is the difference
between DNA damage and
mutation??
It is important to distinguish between DNA
damage and mutation, the two major types of
error in DNA.
DNA damage and mutation are fundamentally
different.
Damage is a physical abnormality in the DNA, such
as single and double strand breaks, 8hydroxydeoxyguanosine residues and polycyclic
aromatic hydrocarbon adducts.
In contrast to DNA damage, a mutation is a
change in the base sequence of the DNA. A
mutation cannot be recognized by enzymes once the
base change is present in both DNA strands, and
thus a mutation cannot be repaired.
At the cellular level, mutations can cause
alterations in protein function and regulation.
Mutations are replicated when the cell replicates.
In a population of cells, mutant cells will increase or
decrease in frequency according to the effects of
the mutation on the ability of the cell to survive
and reproduce.
What are the types of DNA
damage??
1. Base loss
2. Base modification
3. Replication errors
4. Inter-strand X-links
5. DNA-protein X-links
6. Strand Breaks
1. Base loss
The bond linking DNA bases with
deoxyribose is labile under physiological
conditions.
Within a typical mammalian cell, several
thousand purines and several hundred
pyrimidines are spontaneously lost per
diploid genome per day.
Loss of a purine or pyrimidine base creates
an apurinic/apyrimidinic (AP) site (also
called an abasic site):
2. Base modification
2a. Deamination
The primary amino groups of nucleic acid
bases are somewhat unstable. The amino
group is removed from the amino acid and
converted to ammonia.
In a typical mammalian cell, about 100 uracils
are generated per haploid genome per day
in this fashion.
Other deamination reactions include
conversion of adenine to hypoxanthine,
guanine to xanthine, and 5-methyl cytosine
to thymine.
Example: cytosine deamination
Spontaneous deamination is the hydrolysis reaction
of cytosine into uracil, releasing ammonia in the
process.
2. Base modification
2b. Chemical modification
The nucleic acid bases are susceptible to
numerous modifications by a wide variety
of chemical agents.
For example, several types of hyperreactive oxygen (singlet oxygen, peroxide
radicals, hydrogen peroxide and hydroxyl
radicals) are generated as byproducts
during normal oxidative metabolism and
also by ionizing radiation (X-rays, gamma
rays). These are frequently called
Reactive Oxygen Species (ROS). ROS can
modify DNA bases. A common product of
thymine oxidation is thymine glycol:
Another type of chemical modification:
methylation/alkylation
• Many environmental chemicals, including "natural"
ones (frequently in the food we eat) can also
modify DNA bases, frequently by addition of a
methyl or other alkyl group (alkylation).
• In addition, normal metabolism frequently leads to
alkylation.
• It has been shown that S-adenosylmethionine, the
normal biological methyl group donor, reacts
accidentally with DNA to produce alkylated bases
like 3-methyladenine at a rate of several thousand
per day per mammalian diploid genome.
2. Base modification
2b. Photodamage
Ultraviolet light is absorbed by the
nucleic acid bases, and the resulting
influx of energy can induce chemical
changes.
• The most frequent photoproducts are
the consequences of bond formation
between adjacent pyrimidines within
one strand, and, of these, the most
frequent are cyclobutane pyrimidine
dimers (CPDs).
Ultraviolet light
induces the
formation of
covalent linkages
by reactions
localized on the
C=C double bonds
3. Replication errors
Another major source of potential
alterations in DNA is the generation of
mismatches or small insertions or deletions
during DNA replication.
Although DNA polymerases are moderately
accurate, and most of their mistakes are
immediately corrected by polymeraseassociated proofreading exonucleases,
nevertheless the replication machinery is
not perfect.
4. Inter-strand crosslinks
By attaching to bases on both strands,
bifunctional alkylating agents such as
the psoralens can cross-link both
strands.
Cross-links can also be generated by UV
and ionizing radiation.
5. DNA-protein crosslinks
DNA topoisomerases generate covalent links
between themselves and their DNA
substrates during the course of their
enzymatic action.
Usually these crosslinks are transient and
are reversed as the topoisomerase action
is completed. Occasionally something
interferes with reversal, and a stable
topoisomerase-DNA bond is established.
Bifunctional alkylating agents and radiation
can also create crosslinks between DNA
and protein molecules. All of these lesions
must be repaired.
6. Strand breaks
Single-strand and double-strand breaks
are produced at low frequency during
normal DNA metabolism by
topoisomerases, nucleases, replication
fork "collapse", and repair processes.
Breaks are also produced by ionizing
radiation.
What can the cell do to
protect itself?
DNA damage recognition
DNA damage is recognized by sensor proteins
that then initiate a network of signal transduction
pathways.
This ultimately results in the activation of
effector proteins that execute the functions of
the DNA damage response, including recruitment
of DNA repair proteins, cell cycle arrest, damage
induced transcription, or the induction of
apopotosis.
An option: DNA damage
checkpoints
• After DNA damage, cell cycle checkpoints are
activated.
• Checkpoint activation pauses the cell cycle and
gives the cell time to repair the damage before
continuing to divide.
Cell Cycle
• The cell cycle of eukaryotic cells can be divided
into four successive phases:
• M phase (mitosis), in which the nucleus and the
cytoplasm divide;
• S phase (DNA synthesis), in which the DNA in
the nucleus is replicated,
• two gap phases, G1 and G2.
• The G1 phase is a critical stage, allowing
responses to extracellular cues that induce
either commitment to a further round of cell
division or withdrawal from the cell cycle (G0) to
embark on a differentiation pathway.
The transition
from one phase of
the cell cycle to
the next is
controlled by
cyclin–CDK
(cyclin-dependent
kinase) complexes
which ensure that
all phases of the
cell cycle are
executed in the
correct order.
• DNA damage checkpoints occur at the
G1/S and G2/M boundaries. An intra-S
checkpoint also exists.
• Checkpoint activation is controlled by two
master kinases, ATM and ATR.
• ATM responds to DNA double-strand
breaks and disruptions in chromatin
structure, whereas ATR primarily responds
to stalled replication forks.
• These kinases phosphorylate downstream
targets in a signal transduction cascade,
eventually leading to cell cycle arrest.
DNA damage checkpoints
• A class of checkpoint mediator proteins including
BRCA1, MDC1, and 53BP1 has also been identified.
• These proteins seem to be required for
transmitting the checkpoint activation signal to
downstream proteins.
• p53 is an important downstream target of ATM
and ATR, as it is required for inducing apoptosis
following DNA damage.
What happens if we have
defective ATM??
ataxia telangiectasia mutated
Disease: Ataxia-telangiectasia
Ataxia-telangiectasia is a rare, childhood
neurological disorder that causes degeneration in
the part of the brain that controls motor
movements and speech.
Its most unusual symptom is an acute sensitivity to
ionizing radiation, such as X-rays or gamma-rays.
The first signs of the disease, which include
delayed development of motor skills, poor balance,
and slurred speech, usually occur during the first
decade of life.
Telangiectasias (tiny, red "spider" veins), which
appear in the corners of the eyes or on the
surface of the ears and cheeks, are characteristic
of the disease, but are not always present and
generally do not appear in the first years of life.
About 20% of those with A-T develop cancer,
most frequently acute lymphocytic leukemia or
lymphoma.
Many individuals with A-T have a weakened
immune system, making them susceptible to
recurrent respiratory infections.
ATM mutations are associated
with breast cancer
• Researchers have found that having a
mutation in one copy of the ATM gene in
each cell (particularly in people who have at
least one family member with ataxiatelangiectasia) is associated with an
increased risk of developing breast cancer.
• About 1 percent of the United States
population carries one mutated copy of the
ATM gene in each cell. These genetic
changes prevent many of the body's cells
from correctly repairing damaged DNA.
So thank goodness for
DNA Repair
DNA repair refers to a collection of processes by
which a cell identifies and corrects damage to the
DNA molecules that encode its genome.
In human cells, both normal metabolic activities
and environmental factors such as UV light and
Radiation can cause DNA damage, resulting in as
many as 1 million individual molecular lesions per
cell per day.
Many of these lesions cause structural damage to
the DNA molecule and can alter or eliminate the
cell's ability to transcribe the gene that the
affected DNA encodes.
DNA repair mechanisms
Cells cannot function if DNA damage corrupts the
integrity and accessibility of essential information in
the genome (but cells remain superficially functional
when so-called "non-essential" genes are missing or
damaged).
Depending on the type of damage inflicted on the
DNA's double helical structure, a variety of repair
strategies have evolved to restore lost information.
Direct reversal
Cells are known to eliminate damage to their DNA
by chemically reversing it.
These mechanisms do not require a template, since
the types of damage they counteract can only occur
in one of the four bases.
Such direct reversal mechanisms are specific to
the type of damage incurred and do not involve
breakage of the phosphodiester backbone.
An example:
methylation of guanine bases, is directly reversed
by the protein methyl guanine methyl transferase
(MGMT), the bacterial equivalent of which is called
as ogt.
This is an expensive process because each MGMT
molecule can only be used once; that is, the reaction
is stoichiometric rather than catalytic.
How do we end up with
methylated Guanine???
Exposure to alkylating agents!
Damage induced mimics
some chemotherapeutics
Damage induced mimics
environmental exposures
guanine
X
cytosine
thymine
O6-meG can mispair with thymine
G/C to A/T transitions
Can be cytotoxic or mutagenic lesio
Using genomics to PREDICT population
responses to exposures
Can we use gene expression levels
to predict responses to
DNA alkylating agents?
Fry et al, Genes and Development 2008
NIH PDR Cell Lines Represent Healthy
Genetically Diverse Population
Native
Native(30)
(30)
Mexican
Mexican
(60)
European
European
(120)
(120)
Asian
Asian(120)
(120)
African
African(120)
(120)
(60)
450 healthy, unrelated individuals
24 lymphoblastoid cell lines
Establish Range of Sensitivity in Cells Exposed
to MNNG (0.5 ug/ml)
Sensitive
Resistant
Training Population
Establish training population based on extreme
responders
Sensitive
Resistant
Training Population
Alkylation-Sensitivity-Associated Gene Sets
Identified
Sensitive
Resistant
Expression intensity
Sensitive
Resistant
250
200
150
100
50
0
0
20
40
60
80
100
% Control growth
Statistically Significant
Differential Expression
1.5 FC , p-value < 0.05
Statistically significant
association (p<0.01) of %
control growth and
expression
Apply two-class prediction algorithm:
SVM to 16 cell lines of test population
Sensitivity
94% accuracy
6
4
9 20 12
8
22 7
Basal
Treated
Ratio
48 genes 121 genes 39 genes
Resistance
-1
0
+1
High expression in
MNNG resistant cells
6
Sensitive
4
9
20 12
Resistant
8
22
7
2 genes
48 genes
Low
-1
0
High
+1
The top hit: high expression in
MNNG resistant cells
Resistant
Sensitive
The most significant positive association of
MGMT expression with resistance
active
inactive
in
MGMT
1. Base excision repair (BER), which repairs
damage to a single base caused by oxidation,
alkylation, hydrolysis, or deamination.
The damaged base is removed by a DNA
glycosylase, resynthesized by a DNA
polymerase, and a DNA ligase performs the final
nick-sealing step.
These hydrolyze the Nglycosylic bond between
the base and the
deoxyribose, as
illustrated here by the
action of uracil DNA Nglycosylase:
2. Nucleotide excision repair (NER), which
recognizes bulky, helix-distorting lesions such as
pyrimidine dimers and 6,4 photoproducts.
A specialized form of NER known as transcriptioncoupled repair deploys NER enzymes to genes that
are being actively transcribed.
NER involves the following
steps:
• Damage recognition
• Binding of a multi-protein complex at the damaged
site
• Double incision of the damaged strand several
nucleotides away from the damaged site, on both
the 5' and 3' sides
• Removal of the damage-containing oligonucleotide
from between the two nicks
• Filling in of the resulting gap by a DNA polymerase
• Ligation
What happens if we have
defective NER??
Xeroderma pigmentosum (XP)
Xeroderma pigmentosa, or XP,
is an autosommal ressessive
genetic disorder of DNA
repair in which the ability to
repair damage caused by
ultraviolet (UV) light is
deficient (NER deficiency).
This disorder leads to
multiple basal cell carcinomas
(basaliomas) and other skin
malignancies at a young age.
In severe cases, it is necessary to avoid
sunlight completely.
The two most common causes of death for
XP victims are metastatic malignant
melanoma and squamous cell carcinoma.
Cockayne syndrome is a rare autosomal
recessive congenital disorder characterized by
growth failure, impaired development of the
nervous system, abnormal sensitivity to sunlight
(photosensitivity), and premature aging.
3. Mismatch repair (MMR), which corrects
errors of DNA replication and recombination
that result in mispaired (but undamaged)
nucleotides.
Are there health effects
from MMR deficiency?
Double-strand breaks
Double-strand breaks (DSBs), in which both
strands in the double helix are severed, are
particularly hazardous to the cell because they
can lead to genome rearrangements.
Various mechanisms exist to repair DSBs:
non-homologous end joining (NHEJ),
recombinational repair (also known as templateassisted repair or homologous recombination
repair
In NHEJ
DNA Ligase IV, a
specialized DNA Ligase
that forms a complex
with the cofactor
XRCC4, directly joins the
two ends.
DNA ligase, shown
above repairing
chromosomal damage,
is an enzyme that
joins broken
nucleotides together
by catalyzing the
formation of an
internucleotide ester
bond between the
phosphate backbone
and the deoxyribose
nucleotides.
Recombinational Repair
Recombinational repair requires the presence of
an identical or nearly identical sequence to be
used as a template for repair of the break.
The enzymatic machinery responsible for this
repair process is nearly identical to the
machinery responsible for chromosomal
crossover during meiosis.
This pathway allows a damaged chromosome to be
repaired using a sister chromatid (available in G2
after DNA replication) or a homologous
chromosome as a template.
Translesion synthesis
Translesion synthesis is a DNA damage tolerance
process that allows the DNA replication machinery
to replicate past DNA lesions such as thymine
dimers or AP sites.
It involves switching out regular DNA polymerases
for specialized translesion polymerases (e.g. DNA
polymerase V), often with larger active sites that
can facilitate the insertion of bases opposite
damaged nucleotides.
The polymerase switching is
thought to be mediated by, among
other factors, the posttranslational modification of the
replication processivity factor
PCNA.
Translesion synthesis polymerases
often have low fidelity (high
propensity to insert wrong bases)
relative to regular polymerases.
PCNA
Proliferating Cell Nuclear Antigen
DNA repair and cancer
Inherited mutations that affect DNA repair genes
are strongly associated with high cancer risks in
humans.
Hereditary nonpolyposis colorectal cancer (HNPCC)
is strongly associated with specific mutations in the
DNA mismatch repair pathway.
BRCA1 and BRCA2, two famous mutations conferring
a hugely increased risk of breast cancer on carriers,
are both associated with a large number of DNA
repair pathways, especially NHEJ and homologous
recombination.
Modern cancer treatments attempt to localize
the DNA damage to cells and tissues only
associated with cancer, either by physical means
(concentrating the therapeutic agent in the
region of the tumor) or by biochemical means
(exploiting a feature unique to cancer cells in the
body).
Cancer Chemotherapy
The hallmark of all cancers is continuous cell division.
Each division requires both
the replication of the cell's DNA (in S phase) and
transcription and translation of many genes needed
for continued growth.
So, any chemical that damages DNA has the potential
to inhibit the spread of a cancer.
Many (but not all) drugs used for cancer therapy do
their work by damaging DNA.
The table lists (by trade name as well as generic name)
some of the anticancer drugs that specifically target
DNA.
Cyclophosphamide
Cytoxan®
Melphalan
Alkeran®
Busulfan
Myleran®
Chlorambucil
Leukeran®
Mitomycin
Mutamycin®
Cisplatin
Platinol®
forms crosslinks
Bleomycin
Blenoxane®
cuts DNA strands between GT or GC
Irinotecan
Camptosar®
Mitoxantrone
Novantrone®
inhibit the proper functioning of enzymes (topoisomerases) needed to
unwind DNA for replication and transcription
Dactinomycin
Cosmegen®
inserts into the double helix preventing its unwinding
alkylating agents; form interstrand and/or intrastrand crosslinks
Some questions
• How many DNA repair proteins are
there in humans?
• What about conservation across
species??