Download Section D - Prokaryotic and Eukaryotic Chromosome Structure

Section F - DNA Damage,
repair and recombination
Contents
F1 Mutagenesis
Mutation, Replication fidelity, Physical mutagens,
Chemical mutagens, Direct mutagenesis, Indirect
mutagenesis
F2 DNA damage
DNA lesions, Oxidative damage, Alkylation, Bulky adducts
F3 DNA repair
Photoreactivation, Alkyltransferase, Excision repair,
Mismatch repair, Hereditary repair defects
F4 Recombination
Homologous recombination, Site-specific recombination,
Transposition
F1 Mutagenesis —
Mutation
Mutation ：Permanent, heritable( 可遗传的)
alterations in the base sequence of DNA.
Reasons
1. Spontaneous errors in DNA replication or
meiotic recombination.
2. A consequence of the damaging effects of
physical or chemical mutagens on DNA.
Point mutation
(a single base change)
Transition (转换) : Purine or pyrimidine is
replaced by the other. AG T C
Transversion(颠换): a purine is replaced by
a pyrimidine or vice verse.
A T or C
T  A or G
G T or C
C  A or G
Effects of a point mutation
•Noncoding DNA
•Nonregulatory DNA
•3rd position of a codon
Phenotypic
effects
Silent mutation
Coding DNA  altered AA Missense
mutation
No
Yes or No
Coding DNA  stop codon
Nonsense mutation
 truncated protein
Yes
Insertions or deletions
The addition or loss of one or more bases in a DNA region
Frameshift mutations
The ORF of a protein encoded gene is changed so
that the C-terminal side of the mutation is
completely changed.
Examples of deletion mutations
Illustrations of five types of
chromosomal mutations.
F1 Mutagenesis —
Replication fidelity
Important for preserve the genetic
information from one generation to the
next.
• Spontaneous errors in DNA replication is very
rare, one error per 1010 base in E. coli.
Molecular mechanisms for the
replication fidelity
1. DNA polymerase: Watson-Crick base pairing
2. 3’ 5’ proofreading exonuclease.
3. RNA priming: proofreading the 5’ end of the lagging strand
4. Mismatch repair (F3)
Proofreading
by
E. coli polymerase
F1 Mutagenesis —
Physical mutagens
• High-energy ionizing radiation: X-rays and γrays  strand breaks and base/sugar
destruction
• Nonionizing radiation : UV light pyrimidine
dimers
F1 Mutagenesis —
Chemical mutagens
Chemical mutagens:
•
Base analogs: direct mutagenesis
•
Nitrous acid: deaminates C to produce U
•
Alkylating agents
•
Arylating agents
F1 Mutagenesis —
Direct mutagenesis
Direct mutagenesis
The stable, unrepaired base with altered base
pairing properties in the DNA is fixed to a mutation
during DNA replication.
OH
H
Br
:G
O
enol form
H
AGCTTCCTA
TCGAAGGAT
Br
O
Keto form
5-BrU
AGCTTCCTA
TCGAAGGAT
1. Base analog
incorporation
AGCTBCCTA
TCGAAGGAT
2. 1st round
of replication
:A
AGCTBCCTA
TCGAGGGAT
3. 2nd round
of replication
AGCTBCCTA
TCGAAGGAT
AGCTCCCTA
TCGAGGGAT
A·TG·C transition
F1 Mutagenesis —
Indirect mutagenesis
Indirect mutagenesis
The mutation is introduced as a result of an
error-prone repair.
•
Translation DNA synthesis to maintain the
DNA integrity but not the sequence accuracy:
when damage occurs immediately ahead of an
advancing fork, which is unsuitable for
recombination repair (F4), the daughter strand is
synthesized regardless of the the base identity
of the damaged sites of the parental DNA.
•
E. coli translession ? replication: SOS response:
Higher levels of DNA damage effectively inhibit
DNA replication and trigger a stress response
in the cell, involving a regulated increase
(induction) in the levels of a number of
proteins. This is called the SOS response.
1. Some of the induced proteins, such as the
UvrA and UvrB proteins, have roles in normal
DNA repair pathways.
2. A number of the induced proteins, however,
are part of a specialized replication system
that can REPLICATE PAST the DNA lesions
that block DNA polymerase III.
Proper base pairing is often impossible and
not strictly required at the site of a lesion
because of the SOS response proteins,
this translesion replication is error-prone.
The resulting increase in mutagenesis does
not contradict the general principle that replication
accuracy is important (the resulting mutations
actually kill many cells). This is the biological price
that is paid, however, to overcome the general
barrier to replication and permit at least a few
mutant cells to survive.
F2 DNA damage —
Oxidative
damage
(氧化损伤）
DNA lesions
DNA lesions
(DNA损害）
Bulky adducts
(加合物）
UV light
1. Occurs under
the normal
conditions
2. Increased by
ionizing radiation
(physical mutagens)
(physical mutagens)
Alkylation
(烷基化作用）
Alkylating agents
(Chemical mutagens)
Carcinogen
(Chemical mutagens)
The biological effect of the
unrepaired DNA lesions
Physical distortion of
the local DNA structure
Blocks replication
and/or transcription
Altered chemistry of the bases
Allowed to remained in the DNA
Living cell
Lethal
(cell death)
A mutation could become fixed
by direct or indirect mutagenesis
Mutagenic
DNA damage and repair
Mutagen (诱变剂）
minor or
moderate
chemical reactivity
of the bases
DNA damage
(lesions)
Error-free
Repairing
Completely repaired
Direct
mutagenesis
Extensive, right
before Replication
Fork (not repairable)
Indirect
mutagenesis
mutations
•Chemical reactivity of bases is
responsible for some DNA lesion
Cytosine deamination and repair
deamination
--ATGCTACG---TACGATGC--
--ATGUTACG---TACGATGC-Uracil DNA glycosylase
U
--ATGCTACG---TACGATGC--
--ATG TACG---TACGATGC--
F2 DNA damage —
Oxidative damage
DNA lesions caused by reactive
oxygen species such as superoxide
and hydroxyl radicals
1. occurs under NORMAL conditions in all aerobic cells
due to the presence of reactive oxygen species (ROS),
such as superoxide, hydrogen peroxide, and the
hydroxyl radicals (-OH).
2. The level of this damage can be INCREEASED by
hydroxyl radicals from the radiolysis of H2O caused
by ionizing radiation
Oxidation products
F2 DNA damage —
Alkylation
Nucleotide modification caused by electrophilic
alkylating agents such as methylmethane sulfonate （甲
基甲烷磺酸盐）and ethylnitrosourea (乙基亚硝基脲)
1. Electrophilic chemicals adds alkyl groups to
various positions on nucleic acids
2. Distinct from those methylated by normal
methylating enzymes.
alkylating agents
Alkylated bases
F2 DNA damage —
Bulky adducts
DNA lesions that distort the double helix
and cause localized denaturation, for
example pyrimidine dimers and arylating
agents adducts
These lesions disrupt the normal function
of the DNA
Cyclobutane pyrimidine dimer(嘧啶二聚体)
Guanine adduct of benzo[a]pyrene
Aromatic
arylating agents
Covalent adducts
F3 DNA repair —
Photoreactivation
Monomerization of
cyclobutane
pyrimidine dimers by
DNA photolyases in
the presence of
visible light
Direct reversal of a lesion and is error-free
F3 DNA repair —
Alkyltransferase (烷基
转移酶)
Removes the alkyl group from
mutagenic O6-alkylguanine which can
base-pair with T. The alkyl group is
transferred to the protein itself and
inactivate it.
Direct reversal of a lesion and is error-free
The response is adaptive because it is
induced in E. coli by low levels of
alkylating agents and gives increased
protection against the lethal and mutagenic
effects of the high doses
F3 DNA repair —
Excision repair
1. Includs nucleotide excision repair
(NER) and base excision repair
(BER).
2. Is a ubiquitous mechanism
repairing a variety of lesions.
3. Error-free repair
Nucleotide excision repair
1. An endonuclease
cleaves DNA a
precise number of
bases on both sides
of the lesions
(UvrABC
endonulcease
removes pyrimidine
dimers)
2. Excised lesion-DNA
fragment is removed
3. The gap is filled by
DNA polymerase I
and sealed by ligase
Base excision repair
DNA glycolases
AP endonuclease
DNA polymerase
DNA ligase
cleaves N-glycosylic bond
cleaves apurinic
or
pyrimidine site
3’5’ cleavage and
& 5’3’ synthesis
F3 DNA repair —
Mismatch repair
A specialized form of excision repair
which deals with any base mispairs
produced during replication and
which have escaped proofreading
error-free
The parental strand is methylated at N6
position of all As in GATC sites, but
methylation of the daughter strand lag a
few minutes after replication
MutH/MutS recognize the mismatched
base pair and the nearby GATC
DNA helicase II, SSB, exonuclease I
remove the DNA fragment including the
mismatch
DNA polymerase III & DNA
ligase fill in the gap
Expensive to keep the accuracy
F3 DNA repair —
Hereditary repair defects
• Xeroderma pigmentosa,
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.
•Xeroderma pigmentosum has an autosomal
recessive pattern of inheritance.
• The most common defect in xeroderma pigmentosum is
an autosomal recessive genetic defect whereby
nucleotide excision repair (NER) enzymes are mutated,
leading to a reduction in or elimination of NER.
• 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.
F4 Recombination —
Homologous recombination
The exchange of homologous regions between
two DNA moleculs
Diploid eukaryotes: crossing over
Haploid prokaryotes:
recA-dependent, Holliday model DNA repair
in replication fork
Diploid eukaryotes: crossing over
1. Homologous chromosomes line up in meiosis (when)
2. The nonsister chromatids exchange equivalent
sections (what)
Haploid prokaryotes recombination
Between the two homologous DNA duplex (where)
1. between the replicated portions of a partially
duplicated DNA
2. between the chromosomal DNA and acquired
“foreign” DNA
Holliday model (How)
recA-dependent bacterial homologous recombination
1. Homologous DNA
pairs
2. Nicks made near Chi
(GCTGGTGG) sites by
a nuclease.
3. ssDNA carrying the 5’
ends of the nicks is
coated by RecA to
form RecA-ssDNA
dilaments.
5’
3’
5’
3’
3’
5’
3’
5’
3. RecA-ssDNA filaments
search the opposite
DNA duplex for
corresponding
sequence (invasion).
4. form a fourbranched Holliday
structure
5. Branch
migration
6. Resolving Holliday junction
RuvAB is an asymmetric complex that
promotes branch migration of a Holliday
junction.
Recombination based
DNA repair at
replication fork
a. Replication encounters
a DNA lesion
b. Skip the lesion & reinitiate on the side of
the lesion
c. Fill the daughter strand
gap by replacing it with
the corresponding
section from the
parental sister strand
d. post-replication repair
of the left lesion
F4 Recombination —
Site-specific recombination
1.Exchange of non-homologous but
specific pieces of DNA (what)
2.Mediated by proteins that
recognize specific DNA
sequences. (how)
Site-specific recombination:
bacteriophage l insertion
1. l-encoded integrase (Int): makes staggered cuts in
the specific sites
2. Int and IHF (integration host factor encoded by
bacteria): recombination and insertion
3. l-encoded excisionase (XIS): excision of the phage
DNA
Site-specific recombination:
Antibody diversity
H and L are all encoded by
three gene segments: V, D, J
V
Two heavy 250
chains (H)
Two light
chains (L)
250
D
J
15
5
4
Enormous number (>108) of different H and L gene
sequences can be produced by such a recombination
F4 Recombination —
Transposition
1. Requires no homology between
sequences nor site-specific
2. Relatively inefficient
3. Require transposase encoded by the
transposon （转座 子）
Various transposons:
In E. coli:
• IS elements/insertion sequence, 1-2 kb,
comprise a transposase gene flanked by a short
inverted terminal repeats
• Tn transposon series carry transposition
elements and b-lactamase (penicillin resistance)
Eukaryotic transposons, many are
retrotransposons:
Yeast Ty element encodes a protein similar to RT
(reverse transcriptase)
Simplified
Transposition
process
Multiple choice questions
1. Per nucleotide incorporated, the spontaneous
mutation frequency in E. coli is
.
A 1 in 106.
B 1 in 108.
C 1 in 109.
D 1 in 1010.
2. The action of hydroxyl radicals on DNA generates a
significant amount of
.
A pyrimidine dimmers.
B 8-oxoguanine.
C O6- methylguanine.
D 7-hydroxymethylguanine.
3. In methyl-directed mismatch repair in E. coli, the
daughter strand containing the mismatched base is
nicked by
.
A MutH endonuclease.
B UvrABC endonuclease.
C AP endonuclease.
D 3' to 5' exonuclease.
4. Illegitimate recombination is another name for
.
A site-specific recombination.
B transposition.
C homologous recombination.
D translesion DNA synthesis.
5. The excision repair of UV-induced
DNA damage is defective in individuals
suffering from
.
A hereditary nonpolyposis colon cancer.
B Crohn's disease.
C classical xeroderma pigmentosum.
D xeroderma pigmentosum variant.
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