Transcript Ch. 14. Mutations and Repair

sickle-cell anemia
Analogy of the effects of substitution, deletion, and insertion of one letter
in a sentence composed of three-letter words, demonstrating point and
frameshift mutations.
Standard base-pairing relationships (a), compared with
anomalous base-pairing that occurs as a result of tautomeric
shifts (b). The long triangle indicates the point at which the base
bonds to the pentose sugar.
NORMAL
RARE FORM
The standard basepairing relationships
compared with two
anomalous
arrangements
occurring as a result of
tautomeric shifts.
The long triangle
indicates the point of
bonding to deoxyribose
Standard base-pairing relationships (a), compared with
anomalous base-pairing that occurs as a result of tautomeric
shifts (b). The long triangle indicates the point at which the base
bonds to the pentose sugar.
A : T to G : C
transition mutation
as a result of a
tautomeric shift in
adenine.
A : T to G : C
transition mutation
as a result of a
tautomeric shift in
adenine.
Tautomeric Shift in Adenine
Formation of a T-double bond-A to a Ctriple bond-G transition mutation as a
result of a tautomeric shift in adenine.
Formation of a T-double bond-A to a Ctriple bond-G transition mutation as a
result of a tautomeric shift in adenine.
MUTAGENS: BASE ANALOGS
5-bromouracil (5-BU) in similar in structure to thymine.
In the common keto form, 5-BU base-pairs normally with
adenine, behaving as an analog of T.
In the rare enol form, it pairs anomalously with guanine,
behaving as an analog of C.
In the common keto form, 5-BU basepairs normally with adenine, behaving as
an analog of T.
In the rare enol form, 5BU pairs anomalously
with guanine, behaving as an analog of C.
ALKYLATION [another way of possible chemical
modifications of bases]
Conversion of guanine (G) to 6-ethylguanine by the
alkylating agent ethylmethane sulfonate (EMS): addition
of ethyl on 6th carbon of the base.
The 6-ethylguanine, being an analog of A. base-pairs with
thymine
MUTAGENIC RADIATION
GOOD NEWS:
MUTATIONS CAN
BE REPAIRED
Direct correction in replication:
DNA polymerase proofreads
in 3’ → 5’ direction
• Synthesis of DNA strand has high rate of
misincorporations (ERRORS), about 1 in
100,000 nucleotides! (10-5); with
proofreading ~ 99% repaired; still 10-7 with
• Mutations in gene that codes for the
proofreading subunit → no
proofreading!“mutator” gene of E. coli
Mismatch repair (after
proofreading, still during
replication)
AAACCTTGGG
TTTGGATCCC
• But how to decide which strand is correct?
• In E. coli, DNA methylation of A on template (in
GATC) strand happens during replication so template is
in fact temporarily ‘highlighted’ by methylase enzyme
• Repair enzyme recognizes mismatch, makes a ‘nick’;
exonuclease degrades a portion; Dna pol + ligase fill the
gap
Post-replication repair
occurs if DNA
replication has skipped
over a lesion, such as a
thymine dimer
(produced by UV).
Through the process of
recombination, the
correct complementary
sequence is recruited
from the parental strand
and inserted into the gap
opposite the lesion. The
new gap that is created is
filled by DNA
polymerase and DNA
ligase.
DNA ligase working on repair
Damaged DNA repaired
by photoreactivation
repair. The bond creating
the thymine dimer is
cleaved by the
photoreactivation
enzyme (PRE), which
must be activated by
blue light in the visible
spectrum.
Base-excision repair (BER) accomplished by uracil DNA glycosylase, AP endonuclease, DNA
polymerase, and DNA ligase. Uracil is recognized as a non-complementary base, excised, and
replaced with the complementary base (C).
Nucleotide-excision repair
(NER) of a UV-induced
thymine dimer. In
actuality, 13 nucleotides
are excised in prokaryotes
and 28 nucleotides are
excised in eukaryotes
during repair.
Two individuals with xeroderma pigmentosum. The 4-year-old
boy on the left shows marked skin lesions induced by sunlight.
Mottled redness (erythema) and irregular pigment changes in
response to cellular injury are apparent. Two nodular cancers are
present on his nose. The 18-year-old girl on the right has been
carefully protected from sunlight since her diagnosis of
xeroderma pigmentosum in infancy. Several cancers have been
removed and she has worked as a successful model.
Xeroderma pigmentosum, or XP, is an autosomal recessive genetic disorder
of DNA repair in which the ability to repair damage caused by ultraviolet (UV)
light is deficient. This disorder leads to multiple basaliomas and other skin
malignancies at a young age. In severe cases, it is necessary to avoid sunlight
completely.
The most common defect in xeroderma pigmentosum is a genetic defect
whereby nucleotide excision repair (NER) enzymes are mutated, leading to a
reduction in or elimination of NER.[1] Unrepaired damage can lead to
mutations, altering the information of the DNA in individual cells. If mutations
affect important genes, like tumor suppressor genes (e.g. p53) or proto
oncogenes then this disorder may lead to cancer. Patients exhibit elevated risk
of developing cancer.
Normally, damage to DNA in epidermal cells occurs during exposure to UV
light. The absorption of the high energy light leads to the formation of
pyrimidine dimers, namely CPD's (cyclobutane-pyrimidine-dimers) and 64PP's (pyrimidine-6-4-pyrimidone photoproducts). The normal repair process
entails nucleotide excision. The damage is excised by endonucleases, then the
gap is filled by a DNA polymerase and "sealed" by a ligase.
Onset of skin
cancers in
patients with or
without
xeroderma
pigmentosum
(XP),
The Dark Side of the Sun is a 1988
American - Yugoslavian drama film
starring a young Brad Pitt about a
young man in search for a cure for a
dreaded skin disease. It is directed by
Bozidar Nikolic.
The footage for the film was shot in
1988 but due to the outbreak of civil
war it had to be abandoned and much
footage was lost.
It was eventually released officially in
1997.
An insertion sequence (IS), shown in red. The terminal
sequences are perfect inverted repeats of one another.
Site-directed mutagenesis.
A single strand of DNA is
isolated and hybridized with a
synthetic oligonucleotide
containing a triplet altered to
encode an amino acid of
choice. After semiconservative
replication, a different
complementary base pair is
present in one of the new
duplexes. Upon transcription
and translation, a mutant
protein that was “designed” in
the laboratory will be
produced.
Structural organization of a copia transposable element in
Drosophila melanogaster, showing the terminal repeats.