1. Mutations can occur through errors in DNA replication, repair, or recombination which can cause substitutions, insertions or deletions of DNA bases. Environmental mutagens like radiation and chemicals can also directly interact with DNA and cause mutations. 2. Some mutations involve changes to a single DNA base pair, while others are larger scale mutations affecting longer DNA segments. Point mutations may substitute one base for another, while insertions or deletions can disrupt the DNA reading frame. 3. Cells have mechanisms like direct repair and photoreactivation to correct some mutations, but errors in these pathways can also lead to mutations if not repaired properly.

1. MUTATIONS AND DNA
REPAIR MECHANISMS

2. Mutations can occur in a number of ways:
1. Errors can occur during DNA replication,
DNA repair, or DNA recombination which
can lead to base-pair substitutions,
insertions, or deletions, as well as mutations
affecting longer stretches of DNA.
2. Mutagens are chemical or physical agents
that interact with DNA to cause mutations.
3. Physical agents include high-energy
radiation like X-rays and ultraviolet light.
4. Some errors can be corrected by direct
repair, while others are repaired by more
complex mechanisms.

3.  MUTATIONS are changes in the genetic material
of a cell (or virus).
 Some are large-scale mutations in which long
segments of DNA are affected (example:
translocations, duplications, and inversions).
 A chemical change in just one base pair of a gene
causes a spontaneous or point mutation.
 A base-pair substitution is a point mutation that
results in replacement of a pair of complimentary
nucleotides with another nucleotide pair.
 Some base-pair substitutions have little or no
impact on protein function.
 If these occur in gametes or gamete-producing
cells, they may be transmitted to future
generations and cause novel traits or defects.

4.  Silent /synonymous mutations changes a codon but
does not alter the amino acid encoded. Alterations of
nucleotides still indicate the same amino acids because
of redundancy in the genetic code. Such mutations may
still have effects on mRNA stability.
 Nonsynonymous mutations result in an altered
sequence in a polypeptide or functional RNA: one or
more components of the sequence are altered or
eliminated, or an additional sequence is inserted into
the product.
 Transversions (blue): replacement
of a purine by a pyrimidine or that of
a pyrimidine by a purine.
 Transitions – (black ): replacement
of one purine by the other or that of
one pyrimidine by the other.

5.  Missense mutations
are those that still
code for an amino
acid but change the
indicated amino
acid.
 Nonsense
mutations change
an amino acid
codon into a stop
codon, nearly
always leading to a
nonfunctional
protein.

6.  Insertions and deletions
are additions or losses of
nucleotide pairs in a gene.
 These have a disastrous
effect on the resulting
protein more often than
substitutions do.
 Unless these mutations
occur in multiples of three,
they cause a frameshift
mutation.
 All the nucleotides
downstream of the deletion
or insertion will be
improperly grouped into
codons.
 The result will be extensive
missense, ending sooner or
later in nonsense -
premature termination.

7. Mutation class Type of mutation Incidence
Base Comparatively common type of mutation in coding
All types
substitutions DNA but also common in noncoding DNA
Transitions and Transitions are more common than transversions,
transversions especially in mitochondrial DNA
Synonymous substitutions are more common than
Synonymous and
nonsynonymous substitutions in coding DNA;
nonsynonymous
conservative substitutions are more common than
substitutions
non-conservative
Gene conversion-like
Rare except at certain tandemly repeated loci or
events (multiple base
clustered repeats
substitution)
One or a few Very common in noncoding DNA but rare in coding
Insertions
nucleotides DNA where they produce frameshifts
Triplet repeat Rare but can contribute to several disorders,
expansions especially neurological disorders
Rare; can occasionally get large-scale tandem
Other large insertions duplications, and also insertions of transposable
elements
One or a few Very common in noncoding DNA but rare in coding
Deletions
nucleotides DNA where they produce frameshifts
Rare, but often occur at regions containing tandem
Larger deletions
repeats or between interspersed repeats
Rare as constitutional mutations, but can often be
Chromosomal Numerical and
pathogenic. Much more common as somatic
abnormalities structural
mutations and often found in tumor cells

8. 1.Purine bases are lost by spontaneous fission of the base-
sugar link.
2.Cytosines, and occasionally adenines, spontaneously
deaminate to produce uracil and hypoxanthine
respectively.
3.Many chemicals, for example alkylating agents, form
adducts with DNA bases.
4.Ultraviolet light causes adjacent thymines to form a
stable chemical dimer.
5.Ionizing radiation causes single or double-strand breaks.
6.Reactive oxygen species in the cell attack purine and
pyrimidine rings.
7.Mistakes in DNA replication result in incorporation of a
mismatched base.
8.Mistakes in replication or recombination leave strand
breaks in DNA.

9. Chemical Modification Depurination Photodamage thymine
dimer
Chemical Modification by O2 free
Deamination radicals

10. (A) depurination (loss of purine bases)
resulting from cleavage of the bond between the purine bases and
deoxyribose, leaving an apurinic (AP) site in DNA and (B)
deamination (converts cytosine to uracil; adenine to hypoxanthine)

11. is the addition of methyl or ethyl groups to
various positions on the DNA bases. Example: alkylation of
guanine by ethylmethane sulfonate (EMS). At the left is a
normal G-C base pair. Note the free O6 oxygen (red) on the
guanine. EMS donates an ethyl group (blue) to the O6
oxygen, creating O6-ethylguanine (right), which base-pairs
with thymine instead of cytosine. Mustard gas (sulfur
mustard) is the most well-known example because of its
use and consequences observed during World War I. It has
two reactive groups that form intra-chain and inter-chain
cross-links on DNA directly.

12. This lesion can be
repaired by an
enzyme (O6-
methylguanine
methyltransferase)
that transfers the
methyl group from O6-
methylguanine to a
cysteine residue in its
active site, and the
original guanine is
restored. This
reaction is
widespread in both
prokaryotes and
eukaryotes, including
humans.

13. from the sun is carcinogenic and
is a principal cause of skin cancer.
 3 types of ultraviolet radiation (UV) from the sun: UVA
(wavelength 320–380 nm), UVB (wavelength 290–320
nm), and UVC (wavelength 200–290 nm).
 UVC penetrates into the superficial layer of the skin,
UVB penetrates into the basal level of the epidermis,
and UVA penetrates into the more acellular dermis level.
 UVB is the most effective carcinogen because it causes
UV photoproducts.
 Cyclobutane pyrimidine dimers are responsible for at
least 80% of UVB-induced mutations. The precise class
of mutations resulting from pyrimidine dimers is a
unique molecular signature of skin cancer.
 UVA indirectly damages DNA via free radical-mediated
damage. Water is fragmented by UVA, generating
electron-seeking ROS that cause DNA damage
(transversions are characteristic of UVA damage).

14. most common type of DNA damage
caused by UV irradiation. (a) UV light cross-links the
two thymine bases on the top strand. This distorts the
DNA so that these two bases no longer pair with their
adenine partners. (b) The two bonds joining the two
thymines form a 4-membered cyclobutane ring (red).
http://highered.mcgraw-hill.com/olc/dl/120082/micro18.swf

15. UV-induced thymine dimers can
be repaired by
photoreactivation. The enzyme
(photolyase) absorbs visible
light and binds to damaged
DNA. The enzyme breaks the
dimer, and finally dissociates
from the repaired DNA. Repair
of pyrimidine dimers by
photoreactivation is common to
prokaryotic and eukaryotic
cells, including E. coli, yeasts,
and some species of plants and
animals. Photoreactivation is
not universal; many species
(including humans) lack this
mechanism of DNA repair.
15

16.  UV-damaged skin cells are eliminated by initiating apoptosis
(peeling of the skin after a sunburn).
 Mutations in the p53 gene (tumor suppressor p53 protein is
an important regulator of apoptosis). These mutations yield
9 hot spots which are sites where removal of cyclobutane
pyrimidine dimers is particularly slow, and consequently
allows the proliferation of mutated p53 cells.
 UV radiation thus induces the formation of tumor cells by
blocking apoptosis, and clonal expansion of the p53 mutants.
 Sunscreens work on the basis of including UV-absorbing
organic chemicals (e.g. cinnamates), inorganic zinc-
containing pigments, or titanium oxides in their ingredients
to minimize UV absorption by the skin.
 Sunscreens must be used with care since some compounds
may be photosensitized carcinogens, (chemicals that can be
activated by UV to become carcinogenic), e.g. 5-methoxy
psoralen, and fluoroquinolone antibiotics (stay out of the sun
during their administration)!

17. high-energy radiation capable of
producing ionization in substances through which it
passes, e.g. x-rays, alpha and beta rays, and neutrons from
a nuclear reaction.
 It can directly ionize atoms comprising DNA, or
indirectly by the interaction with water molecules
(radiolysis) that generate dangerous reactive oxygen
species (ROS): the hydroxyl radical (–OH), hydrogen
peroxide (H2O2), and the superoxide radical (O–2).
 A free radical reacts very strongly with other molecules
as it seeks to restore a stable configuration of electrons.
A free radical may drift about up to 1010 longer than the
time needed for the initial ionization, increasing the
chance of it disrupting DNA and cause mutations.

18.  Oxidation of DNA is one of the main causes of mutation, and
explains why free radicals produced by radiation exposure
as well as endogeneous cellular reactions (e.g., oxidative
respiration and lipid peroxidation) are such potent
carcinogens.
 Oxidation can produce oxidized bases, e.g., adenine
mispairs with 8-oxoguanine during replication leading to a
G→T transversion mutation.
 The -OH radical removes electrons from any molecule in its
path, turning that molecule into a free radical and so
propagating a chain reaction.
 H2O2 is more dangerous to DNA than the -OH radical. Its
slower reactivity gives it time to travel into the nucleus of a
cell, where it is free to wreak havoc upon DNA.
 The superoxide radical is not very reactive but acts more as
a catalyst for the generation of the other ROS intermediates.
 Double-strand DNA breaks cause ionizing radiation-induced
carcinogenesis.

19. :
 The common mechanism of action is that an electrophilic
(electron-deficient) form reacts with nucleophilic sites (sites
that can donate electrons) in the purine and pyrimidine rings
of nucleic acids.
 Some chemicals are base analogues that may be substituted
into DNA, and pairs incorrectly during DNA replication.
 Other mutagens interfere with DNA replication by inserting
into DNA and distorting the double helix.
 Still others cause chemical changes in bases (DNA adducts)
that change their pairing properties.
Carcinogens can be segregated into 10 groups:
polycyclic aromatic hydrocarbons carbamates
halogenated compounds aromatic amines
nitrosamines and nitrosamides azo dyes
hyrazo and azoxy compounds natural products
inorganic carcinogens
miscellaneous compounds (alkylating agents,
aldehydes, phenolics)

20. ) are
carcinogens produced by cooking
meat, formed from heating amino acids
and proteins. About 20 HCAs have
been identified. Three examples, Phe-
P-1, IQ, and Mel Q, are shown.
These are examples of carcinogens to
which we are exposed daily and which
are produced in our own kitchens!
Oven roasting, marinading, and coating
food with breadcrumbs before frying
are modifications that may reduce the
formation of HCAs.

21. are found in tobacco or are
formed when preservative
nitrites react with amines in fish
and meats during smoking. Their
principal carcinogenic product is
alkylated O6 guanine derivatives.
(a) An example of nitrosamines: alkylnitrosoureas.
(b) A potential carcinogenic product of nitrosamines: O6 adduct of
guanine. Guanine is shown for comparison.

22. treatment of DNA results in the
conversion of adenine into hypoxanthine, which pairs
with cytosine, inducing a transition from A-T to G-C.
induce frameshift mutations by
intercalating into the DNA, leading to the incorporation
of an additional base on the opposite strand.

23. . The compound, produced by molds
that grow on peanuts, is activated by cytochrome
P450 to form a highly reactive species that modifies
bases such as guanine in DNA, leading to mutations.

24.  Asbestos is a group of fibrous
silicate minerals that was used
extensively in building materials
because of its insulating properties
but is now prohibited due to
association with several diseases of
the lung, including lung cancer and
mesothelioma.
 Erionite is a fibrous zeolite mineral
formed from volcanic rock.
 Mechanisms of carcinogenesis
include generation of ROS and
induction of a chronic inflammatory
response. Genetics may predispose
some people to the carcinogenic
effects of fibrous materials.

25. 1. Areas of investigation on the molecular events behind the mechanism
of bacteria-induced transformation include: the promotion of host cell
proliferation, the generation of oxygen free radicals and subsequent
DNA damage, and the activation of oncogenes.
2. DNA tumor viruses encode viral proteins that block tumor suppressor
genes, often by protein–protein interactions. Retroviruses may cause
cancers in animals by encoding mutated forms of normal genes (i.e.
oncogenes) that have a dominant effect in host cells. Examples:
 Human papillomavirus (HPV) - cervical cancer
 Kaposi’s sarcoma-associated herpes virus (KSHV) - Kaposi’s
sarcoma
 Hepatitis B virus - liver cancer
 Epstein–Barr virus (EBV) - nasopharyngeal carcinoma
 Human T-cell leukemia virus type 1 (HTLV-1) – a retrovirus known
to cause acute T-cell leukemia (ATL)
 Helicobacter pylori - a Gram-negative spiral bacterium that
establishes chronic infection and ulcers in the stomach and one of
the causative agents of gastric cancer.
 The typhoid pathogen, Salmonella enterica serovar Typhi (S.
typhi), establishes chronic infection in the gallbladder and has
been linked to hepatobiliary and gallbladder carcinoma.

26. The bases of DNA can exist in rare This base
tautomeric forms. The imino analog of thymine has a
tautomer of adenine can pair with higher tendency to form an
cytosine, eventually leading to a enol tautomer than does
transition from A-T to G-C. thymine itself. The pairing of
(Tautomerization is the the enol tautomer of 5-
interconversion of two isomers that bromouracil with guanine will
differ only in the position of protons
lead to a transition from T-A
and often, double bonds).
to C-G.

27. (a)
(b) Metabolic activation of BP (Benzopyrene)

28. Benzopyrene ( found in cigarette smoke) reacts with DNA
bases, resulting in the addition of large bulky chemical
groups to the DNA molecule and cause G→T transversions.
Locations of these adducts matched the distribution of p53
gene mutations in lung tumors from smokers (Science,1996).
It is estimated that 104 to 106 mutations occur in a single
human cell per day. Each day the DNA of a human cell loses
about 5,000 purines, and about 100 cytosines spontaneously
deaminate to uracil. Damage to DNA can block replication or
transcription, and can result in a high frequency of mutations.

29. Under normal circumstances, the immense error
burden is successfully dealt with by the highly
efficient cellular DNA repair mechanisms.
Major DNA repairing mechanisms: base excision,
nucleotide excision and mismatch repair.

30. A DNA glycosylase specific for G-T
mismatches, usually formed by
deamination of 5-methyl C residues, flips
the thymine base out of the helix and then
cuts it away from the sugar-phosphate
DNA backbone (1), leaving just the
deoxyribose (black dot). An endonuclease
specific for the resultant baseless site then
cuts the DNA backbone (2), and the
deoxyribose phosphate is removed by an
endonuclease associated with DNA
polymerase (3). The gap is then filled in by
DNA Pol ß and sealed by DNA ligase (4),
restoring the original G-C base pair.

31. DNA's bases may be
modified by deamination
or alkylation. The position
of the modified
(damaged) base is called
the "abasic site" or "AP
site". DNA glycosylase
can recognize the AP site
and remove its
base. Then, the AP
endonuclease removes
the AP site and
neighboring
nucleotides. The gap is
filled by DNA polymerase I
and DNA ligase.

32. Proteins UvrA,
UvrB, and UvrC are
involved in removing the
damaged nucleotides
(e.g., the dimer induced
by UV light). The gap is
then filled by DNA
polymerase I and DNA
ligase. In yeast, the
proteins similar to Uvr's
are named RADxx
(radiation), such as
RAD3, RAD10, etc.

33. A DNA lesion that
causes distortion of the double
helix, such as a thymine dimer, is
initially recognized by a complex
of the XP-C (Xeroderma
pigmentosum C protein) and 23B
proteins (1). This complex then
recruits transcription factor TFIIH,
whose helicase subunits, powered
by ATP hydrolysis, partially unwind
the double helix. XP-G and RPA
proteins then bind to the complex
and further unwind and stabilize
the helix until a bubble of ≈25
bases is formed (2). Then XP-G
(now acting as an endonuclease)
and XP-F, a 2nd endonuclease, cut
the damaged strand at points 24–
32 bases apart on each side of the
lesion (3).

34. This releases the DNA fragment with the damaged
bases, which is degraded to mononucleotides.
Finally the gap is filled by DNA polymerase exactly as
in DNA replication, and the remaining nick is sealed by
DNA ligase (4 )

35. The mismatch repair system
detects and excises
mismatched bases in newly
replicated DNA, which is
distinguished from the
parental strand because it has
not yet been methylated. MutS
binds to the mismatched base,
followed by MutL. The binding
of MutL activates MutH, which
cleaves the unmodified strand
opposite a site of methylation.
MutS and MutL, together with
helicase II, SSB proteins, and
an exonuclease, then excise
the portion of the unmodified
strand that contains the
mismatch. The gap is then
filled by DNA polymerase and
sealed by ligase.

36.  Mismatch repair in eukaryotes may be similar to
that in E. coli. Homologs of MutS and MutL
have been identified in yeast, mammals, and
other eukaryotes. MSH1 to MSH5 are
homologous to MutS; MLH1, PMS1 and PMS2
are homologous to MutL.
 Germline mutations of MSH2, PMS1 and PMS2
are related to colon cancer. Loss of function of
the protein products encoded by these genes is
responsible for complete loss of mismatch
repair.
 In eukaryotes, the mechanism to distinguish the
template strand from the new strand is still
unclear, but maybe related to the action of DNA
methylases (the old DNA strand is methylated).

37. A complex of the
MSH2 and MSH6 proteins binds
to a mispaired segment of DNA
such as to distinguish between
the template and newly
synthesized daughter strands
(1). This triggers binding of the
MLH1 endonuclease, as well as
other proteins such as PMS2,
which has been implicated in
onco-genesis through mismatch-
repair mutations. A DNA helicase
unwinds the helix and the
daughter strand is cut; an
exonuclease then removes
several nucleotides, including
the mismatched base (2). Finally,
as with base excision repair, the
gap is then filled in by a DNA
polymerase (Pol, in this case)
and sealed by DNA ligase (3 ).

38. The presence of a thymine
dimer blocks replication, but
DNA polymerase can bypass
the lesion and reinitiate
replication at a new site
downstream of the dimer. The
result is a gap opposite the
dimer in the newly synthesized
DNA strand. In
recombinational repair, this
gap is filled by recombination
with the undamaged parental
strand. Although this leaves a
gap in the previously intact
parental strand, the gap can
be filled by the actions of
polymerase and ligase, using
the intact daughter strand as a
template. Two intact DNA
molecules are thus formed,
and the remaining thymine
dimer eventually can be
removed by excision repair.

39. If the replication fork encounters an
unrepaired lesion or strand break, replication generally halts and the fork may
collapse. A lesion is left behind in an unreplicated, single-stranded segment of
the DNA; a strand break becomes a double-strand break.
There are two possible
avenues for repair:
recombinational DNA
repair or, when lesions
are unusually
numerous, error-prone
repair. The latter
involves DNA
polymerase V,
encoded by the umuC
and umuD genes that
can inaccurately
replicate over many
types of lesions. The
repair mechanism is
referred to as error-
prone because
mutations often result.

40. UV light activates the
RecA co-protease,
which stimulates the
LexA protein (purple)
to cleave itself,
releasing it from the
umuDC operon. This
results in synthesis
of UmuC and UmuD
proteins, which
somehow allow DNA
synthesis across
from a thymine dimer,
even though mistakes
(blue) will be made.

41. The black and
red DNAs represent the
homologous sequences on
sister chromatids. (1) A double-
strand DNA break forms in the
chromatids. (2) The double-
strand break activates the ATM
kinase; this leads to activation
of a set of exonucleases that
remove nucleotides at the break
from the 3’ and 5’ ends of both
broken strands, ultimately
creating single stranded 3’ ends.
In a process that is dependent
on the BRCA1 and BRCA2
proteins, as well as others, the
Rad51 protein (green ovals)
polymerizes on single-stranded
DNA with a free 3’ end to form a
nucleoprotein filament.

42. (3): Aided by yet other
proteins, one Rad52
nucleoprotein filament
searches for the
homologous duplex DNA
sequence on the sister
chromatid, then invades
the duplex to form a joint
molecule in which the
single stranded 3’ end is
base-paired to the
complementary strand on
the homologous DNA
strand. (4) The replicative
DNA polymerases
elongate this 3’ end of the
damaged DNA (green
strand), templated by the
complementary
sequences in the
undamaged homologous
DNA segment.

43. (5) Next this repaired
3’ end of the damaged
DNA pairs with the
single stranded 3’ end
of the other damaged
strand. (6) Any
remaining gaps are
filled in by DNA
polymerase and ligase
(light green),
regenerating a wild-
type double helix in
which an entire
segment (dark and
light green) has been
regenerated from the
homologous segment
of the sister
chromatid.

44.  A double-strand break activates the
ataxia telangiectasia mutated (ATM)
kinase.
 The RAD50/MRE11/NBS1 complex (a
substrate of ATM) uses its 5′–3′
exonuclease activity to create
single-stranded 3′ ends.
 BRCA1/2 aids in the nuclear
transport of RAD51.
 RAD52 facilitates RAD51 binding to
these exposed ends to form a
 nucleoprotein filament.
 RAD51 can exchange a homologous
sequence from a single strand
within a double-stranded molecule
(e.g. a sister chromatid), with a
single-stranded sequence.
 The sequences from the double-
stranded molecule are then used as
a template sequence for repair.
 Resolvases restore the junctions
formed as a result of homologous
recombination, called Holliday
junctions.
 Two copies of intact DNA molecules
are produced with rarely any errors.

45. In general, nucleotide sequences
are butted together that were not
apposed in the unbroken DNA.
These DNA ends are usually from
the same chromosome locus, and
when linked together, several base
pairs are lost. Occasionally, ends
from different chromosomes are
accidentally joined together. A
complex of two proteins, Ku and
DNA-dependent protein kinase,
binds to the ends of a double-strand
break (1). After formation of a
synapse, the ends are further
processed by nucleases, resulting
in removal of a few bases (2), and
the two double-stranded molecules
are ligated together (3). As a result,
the double-strand break is repaired,
but several base pairs at the site of
the break are removed.

47. Several conventional therapies aim to induce extensive DNA
damage in order to trigger apoptosis and paradoxically include
agents classified as carcinogens. Other conventional therapies
inhibit DNA metabolism in order to block DNA synthesis in the
rapidly dividing cancer cells. Still other drugs interfere with the
mechanics of cell division. The development of drug resistance
is a major problem for chemotherapy.
and : have the ability to
form DNA adducts by covalent bonds via an alkyl group or a
platinum atom, e.g. clorambucil and cisplatin. The resulting
DNA damage triggers apoptosis. Cisplatin had a major impact
on ovarian cancer, but associated with irreversible kidney
damage. Carboplatin is a less toxic platinum analog.
: are compounds that are structurally similar to
endogenous molecules (e.g. nitrogenous bases of DNA) and
therefore can mimic their role and inhibit nucleic acid
synthesis (e.g. 5-FU and methotrexate).

48. : Doxorubicin is a fungal anthracycline
antibiotic that inhibits topoisomerase II. The plant
alkaloids vincristine and vinblastine (from the periwinkle
plant) bind to tubulin and prevent microtubule assembly.
Paclitaxel (taxol) binds to the β-tubulin subunit in
polymers and stabilizes the microtubules against
depolymerization. Thus two opposing strategies can be
used to disrupt the mitotic spindle.
Ionizing radiation is delivered to the
tumor by electron linear accelerators. Radiation-induced
damage can become permanent due to the generation
of ROS if oxygen is present. More double-strand breaks
occur in cells irradiated in the presence of oxygen than
in cells irradiated in the absence of oxygen. Targeting of
the tumor has been made more precise by modern
techniques such as magnetic resonance imaging (MRI)
and computed tomography (CT) which produce 3-D
images of the tumor within the body.

49. If DNA can repair itself,
Go ahead, indulge yourself and
enjoy life’s pleasures!
After all, life is short …
But DNA can only do so much for
itself…
Abusing its potentials can cause YOU
and your future generations
major, major problems!

50. http://highered.mc
graw-
hill.com/sites/0072
437316/student_vie
w0/chapter20/anim
ations.html