Transcript Slides PPT

PHAR2811 Dale’s lecture 6
Repair and Mutation
Telomerases as drug targets
Telomerase is active in between 80 and 90%
of all cancers
• Targeting the RNA component with
antisense oligodeoxynucleotides and
RNaseH (see following slides)
• Reverse transcriptase inhibitors; AZT,
dideoxyguanidine
Telomerases as drug targets
Telomerase is active in between 80 and 90%
of all cancers
• Inhibitors of the catalytic protein subunit
• G-quadruplex stabilisers..the 3’ overhang is
G rich and tetra-stranded DNA can form.
This only happens with G-rich sequences.
If this aberrant structure can be stabilised
the end is blocked and telomerase activity
is inhibited.
Oligodeoxynucleotide treatment
5’
3’
3’
TTAGGG
AAUCCCAAU
5’
3’
5’
Telomerase, containing an RNA component.
This enzyme was discovered by an
Australian scientist, Elizabeth Blackburn.
The sequence shown here is the
tetrahymena sequence originally worked on
by Elizabeth Blackburn. The human
sequence is 11 nucleotides, rather than 9.
TTAGGGTTA
5’
TTAGGG
3’
TTAGGGTTA
TTAGGGTTA
TTAGGGTTA
AAUCCCAAU
3’
5’
3’
5’
If oligonucleotides are introduced to the cell
which are complementary to the RNA sequence that binds to
the telomere they will bind to the telomerase preventing it
binding to the telomere.
It is tricky
to
introduce
these to
the cell!
TTAGGGTTA
5’
TTAGGG
3’
TTAGGGTTA
TTAGGGTTA
TTAGGGTTA
AAUCCCAAU
3’
5’
3’
5’
If oligonucleotides are introduced to the cell
which are complementary to the RNA sequence that binds to
the telomere they will bind to the telomerase preventing it
binding to the telomere.
TTAGGGTTA
5’
TTAGGG
3’
TTAGGGTTA
TTAGGGTTA
TTAGGGTTA
3’
5’
3’
5’
If you combine it with RNaseH you will then destroy the
telomerase by degrading the RNA . RNaseH degrades RNA
in DNA:RNA hybrids. Activation of the cell’s endogenous
RNaseH is being considered.
Repair
• Repair of DNA is vital to the survival of the
species.
• DNA has the capability to repair itself,
unlike RNA. The extra copy provides the
template and elaborate repair mechanisms
have evolved to correct corruptions.
• Many errors at the time of replication are
corrected by the 3’  5’ exonuclease
activity of DNA pols I & III.
Repair
• There are corruptions to the sequence which
occur after replication.
• An example. There are 3.2 X 109 purine
nucleotides in the human genome. Each day
~10 000 glycosidic bonds are cleaved from
these purines in a given cell under physiological
conditions.
• The conclusion: your cells contain some nasty
little compounds. There are 130 genes which
encode proteins responsible for repair in the
human genome.
UV damage
(or the battle of the bulge)
• DNA, when exposed to UV irradiation, forms
pyrimidine dimers.
• Carbons 5 and 6 of two consecutive pyrimidines
form covalent bonds with each other (cyclobutyl
ring).
• These cyclobutyl bonds are ~1.6 Å which is
much shorter than the distance between the
bases (~3.4 Å). This causes a bulge in the
double helix which prevents transcription or
replication. This dimer has to go!!
Pyrimidine dimers
UV exposure
UV damage
(or the battle of the bulge)
• There are a couple of ways of repairing
this dimer.
• One is to cleave the bonds directly, with
an enzyme known as photolyase.
• This enzyme uses the energy from visible
light to cleave the cyclobutyl bond.
• Photolyase is present in many prokaryotes
and eukaryotes (not humans however).
Photolyase:
uses energy absorbed by visible light to
break the covalent bond
UV damage
(or the battle of the bulge)
• Another more general type of repair is
excision repair.
• The pyrimidine dimer, and a number of
ajoining nucleotides are cut out by a set of
enzymes responsible for nucleotide
excision repair (NER). NER is carried out
in E. coli by UvrA, UvrB and UvrC.
UV damage
(or the battle of the bulge)
• These enzymes seek out the bulge and
cut out the offending nucleotides.
• The 11 to 12 oligomer is then removed by
UvrD. DNA pol I then comes and fills in the
missing section, using the other strand as
a template.
• The gap is sealed by DNA ligase.
NER
Chunk
excised
Filled in with
DNA pol I
Using its 5’ to
3’polymerase and
its proof reading
3’ exonuclease
activity
3’OH
DNA pol I
nucleotides
Gap sealed with ligase
Uracils in DNA
• Uracil, which comes about from the
spontaneous deamination of cytosine [or
for that matter hypoxanthine, another base
which comes about from the deamination of
adenine and xanthine, derived from the
deamination of guanine], does not belong in
DNA.
• A set of enzymes (base excision repair,
BER) cleaves out the base at the
glycosidic bond leaving an apurinic or
apyrimidinic site (AP).
Uracils in DNA
• The deoxyribose is then excised by an AP
endonuclease along with several ajoining
nucleotides.
• The whole section is then filled in by DNA
pol I and DNA ligase.
Wrong base: corruption
by deamination e.g. cytosine
to uracil, adenine to
hypoxanthine
Base excised
Apurinic or
apyrimidinic
site (AP)
The deoxyribose is
removed by an AP
endonuclease
Extra nucleotides then
removed
DNA pol I and ligase come in to mop up
3’OH
De-purination
• When purine bases are cleaved at the
glycosidic bond (as in the example in the
introduction) the AP endonuclease again
comes into action to remove the
deoxyribose. The DNA pol I and ligase
then mop up.
Mutations
• A mutant gene is one that has a different
sequence to the normal or wild type gene.
• This change is inheritable.
• There are a variety of mutations which
may or may not cause a change in the
phenotype. The vast majority of mutations
are neutral, having no effect (positive or
negative) on the organism.
Mutations
• In multicellular organisms for a mutation to
be inheritable it must be present in the
germline cells (meiotic).
• Mutations in somatic cells (mitotic) ONLY
will not be inherited (cancer).
• Changes to the RNA code (errors in
transcription) are not inherited.
Static Mutations
• Static mutations are those where the
change in the code becomes a stable
incorporation into the genome of the
germline cells as well as all somatic cells
in the organism (except RBC)
• This change is transferred to the next
generation so the genome of the offspring
is the same as the parent.
Static Mutations
• The classic mutations such as sickle cell
anemia, CF, PKU are much studied
examples of this phenomenon.
• The ultimate expression of these
mutations as a phenotype depends on the
genetic information from both parents and
epigenetic factors.
Dynamic Mutations
• Examples here are the trinucleotide
repeats (TNR).
• The mutation increases (increasing
number of repeats) in severity with each
generation
• It also varies between tissues of the same
organism.
Dynamic Mutations
• This leads to genetic anticipation.
• The following generation will be more
affected or the onset of the disorder will be
earlier than the previous generation.
• The increase in the copy number of the
repeat can occur at replication, repair or
recombination – whenever DNA is being
copied.
Dynamic Mutations
• The theories as to the mechanism(s)
causing this type of mutation are many
and varied (which means they won’t be
covered in this course).
• They all involve the formation of an
aberrant loop structure when the DNA
strands are separated. The copying
process somehow stutters then. The more
repeats the more likely the loop.
Types of Mutations
• Transversion: purine replaced by a pyrimidine
or vice versa. This has implications for the 3-D
shape of the helix.
• Transition: pyrimidine for pyrimidine or purine
for purine e.g. AG,CT
• Silent mutations: altered codon still codes for
the same amino acid (code redundancy)
• Frameshift: shifts the reading frame by adding
or deleting base(s). Leads to a non-functional
protein.
Types of Mutations
• Neutral mutation: altered codon codes for a
functional similar amino acid  no effect on the
functionality of protein.
• Point mutation: single base pair change, can
be a substitution, deletion, or addition.
• Missense: altered codon for functionally
different amino acid  these can be lethal
• Nonsense: mutation produces a stop codon 
truncated protein  also dangerous.
Types of Mutations
• Splice mutation: produces a splice site or
removes one (only in eukaryotes)
• Temperature sensitive: mutation causes a
change the protein function which is temperature
sensitive. Usually the protein functions normally
at lower permissible temperatures (<30oC) but is
inactive at higher temperatures (>40oC).
• Reversion: a mutation which reverts to wild
type, referred to as revertants.
• Leaky mutation: A mutation which doesn’t
affect the organism under normal conditions. It
will show up in “stressed” conditions.
Mutagenesis.
• Definition of mutagen: a physical or
chemical agent which causes mutation to
occur at a higher frequency.
• Natural or spontaneous mutations:
These are the mutations which occur at a
normal background rate all the time.
These mutations in the genome can arise
naturally in the course of a cell’s life.
Natural or Spontaneous
Mutagenesis.
• The alternative tautomer of the base. If the
base flips to the alternative tautomer at the time
of replication the wrong nucleotide will pair up
and by two rounds of replication we have a base
pair switch.
• An error in replication which is not picked up
by the proof reading activity of DNA pol III will
result in a mutation.
• Deamination, of cytosines (discussed earlier)
or adenine to hypoxanthine. These can be
corrected by specific repair mechanisms.
• Depurination (see above) can also be
corrected by specific repair mechanisms.
Oxidative deamination of cytosine
NH 2
O
NH3
N
N
deoxyribose
Cytosine
N
O
N
deoxyribose
Uracil
O
Deamination of adenine leads to
base pairing with cytosine
O
NH 2
N
N
N
NH
N
N
Adenine
N
N
Hypoxanthine
Tautomers of Adenine
donor
H
N
H
H
acceptor
N:
donor
acceptor
N
N
N
N
Adenine at pH 7. The ring
N has a pKa of ~4 so at
pH 7 the majority of the
molecules are in this form
H
N
N
N
N
A very small proportion of the
adenines will assume this
tautomeric form at pH 7
Tautomers of Adenine
NH 2
N
N
N:
CH 3
O
HN
N
N
The major
tautomeric form of
Adenine at pH 7
O
Thymine
Tautomers of Adenine
H
:N
N
N
N
H2N
H
N:
N
The very very minor
tautomeric form of
Adenine at pH 7
N
O
This tautomer
bonds to Cytosine
The effect of a tautomeric change
at replication
A-T
A
T
*A
C
A
T
C-G
A-T
T-A
If the adenine (A *A) flips
to the alternative tautomer
at the time of replication,
just as the new incoming
nucleotide is selected it will
base pair to cytosine, rather
than thymine as the donor
and acceptor are the other
way around. Within another
generation you have a GC
instead of an AT.
Induced Mutagenesis.
• Intercalators, planar ring structures which
slide in between the base pairs causing a
disruption to the normal base stacking eg
ethidium Bromide, acridine orange,
actinomycin D.
• Alkylating agents, which methylate or
ethylate bases and result in altered base
pairing during replication e.g
methylmethane sulfonate (MMS),
nitrosamine.
Intercalators
Distance of a base pair, fits in nicely
and separates the base stacking
Induced Mutagenesis.
• Alkylating agents, which methylate or
ethylate bases and result in altered base
pairing during replication e.g
methylmethane sulfonate (MMS),
nitrosamine.
• Anti-cancer drugs, used to treat brain
tumours e.g. Temozolomide or temodal
alkylates guanine residues at positions 6
and 7 and interferes with DNA replication.
Alkylating agents
CH 3
O
N
N
N
NH
N
Guanine
O
alkylating agents
NH 2
N
NH
N
O6-methylguanine
NH 2
Induced Mutagenesis.
• Base altering agents eg nitrous acid
converts amino groups to keto groups.
• Base analogues e.g. 5-Bromouracil which
replace thymine but base pair to G
(tautomeric shift due to Br).
Base analogues
5-Bromouracil which replaces thymine but
base pairs to G (tautomeric shift due to Br).
H
O
O
H3C
Br
NH
N
Thymine
N
O
N
O
5-Bromouracil
Testing Mutagenesis:
the Ames test
• A quick screening test for potential
mutagenic compounds.
• A strain of Salmonella which has a defect
in the histidine biosynthetic pathway is
plated out, as a lawn, on a medium
containing minimal His (just enough to keep
the cells alive but not enough to sustain
proliferation)
Testing Mutagenesis:
the Ames test
• The compound of interest is applied to a
disc in the centre of the plate and the plate
is incubated overnight.
• Different plates with increasing amounts of
the compound are put up.
• Sometimes liver extract is applied also to
check for cellular conversions
Testing Mutagenesis:
the Ames test
• If the compound is mutagenic it will cause
a number of cells to revert to wild type and
grow on the medium; the other cells can’t
because of the His defect.
• The more colonies forming around the disc
the more mutagenic the compound.
• A non-mutagenic compound will have a
few colonies scattered over the whole
plate (spontaneous reversions).
Ames Test
Mutagenic Response
• A mutagenic compound will typically have
a linear dose response.
# colonies
Concentration of potential mutagen
Ames Test
The negative control
A mutagenic compound
9. The Ames test relies on a number of
factors. Which of the following is NOT an
assumption required for the Ames test?
A. Salmonella spontaneously mutate at a very fast
rate
B. Wild type Salmonella can synthesise histidine
de novo (from scratch)
C. Mutagenic compounds will only induce
mutations which cause His reversion
D. Salmonella deficient in histidine synthesis (Sal
his-/-) can be maintained on minimal histidine
medium without proliferation
E. A visible colony on an agar plate is the result of
cell proliferation from a single cell
9. The Ames test relies on a number of
factors. Which of the following is NOT an
assumption required for the Ames test?
A. Salmonella spontaneously mutate at a very fast
rate
B. Wild type Salmonella can synthesise histidine
de novo (from scratch)
C. Mutagenic compounds will only induce
mutations which cause His reversion
D. Salmonella deficient in histidine synthesis (Sal
his-/-) can be maintained on minimal histidine
medium without proliferation
E. A visible colony on an agar plate is the result of
cell proliferation from a single cell