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SECTION F
DNA damage, repair, and recombination
F1
Mutagenesis (诱变）
F2
DNA damage
F3
DNA repair
F4
Recombination
F1 Mutagenesis
Mutation
Replication fidelity
Mutagens: chemical & physical
Mutagenesis: direct & indirect
Replication
Fidelity
(复制的忠实
性）
Mutation
（突变）
Mutagenesis
Mutagens
（诱变剂）
Mutation-1
Concept: Permanent, heritable alterations
in the base sequence of DNA
Arise either through spontaneous errors in DNA
replication or meiotic recombination or as a
consequence of the damaging effects of physical or
chemical agents on the DNA.
Mutation-2
Point mutation: is the simplest mutation-a single
base change.
1-Transition (转换): Purine or pyrimidine is replaced
by the other AG
T C
2-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
Mutation-3
Effects of a point mutation
The sites
Types
Phenotypic
effects
•Noncoding DNA
•Nonregulatory DNA
•3rd position of a codon
Silent mutation
Coding DNA  altered AA
Missense
mutation
Yes or No
Nonsense mutation
Yes
Coding DNA  stop codon
 truncated protein
No
Mutation-4
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 Cterminal side of the mutation is completely changed.
Examples of deletion mutations
Replication fidelity
The need to preserve the meaning of the genetic
information from one generation to the next, so the
error rate of DNA replication is much lower. E.g.
Spontaneous errors in DNA replication is very rare,
one error per 1010 base in E. coli.
Molecular mechanisms of the replication fidelity
1. DNA polymerase: Watson-Crick base pairing
2. The occasional error can be detected and repaired by 3’
5’ proofreading exonuclease.
3. RNA priming: proofreading the 5’ end of the lagging strand
4. Mismatch repair mechanism(F3)
Proofreading
by
E. coli polymerase
(Figure from Gene VII)
Mutagens
The physical and chemical agents of causing DNA damage
that can be converted to mutations.
1- Physical mutagens
High-energy ionizing radiation: x-rays and g-rays  strand
breaks and base/sugar destruction (cause extensive chemical
alterations).
Nonionizing radiation: UV light pyrimidine dimers from
adjacent.
2-Chemical mutagens
Base analogs: direct mutagenesis;
Nitrous acid: deaminates C to produce U and guanine
analog; Alkylating agents and Arylating agents produce
lesions (indirect mutagenesis);
Intercalators: insertion and deletion mutations
Base analogs: derivatives of the normal bases with
altered base pairing properties.
Nitrous acid: deaminates C to produce U, resulting in
G·C  A·U
Mutagenesis
Mutagenesis is the molecular process in
which the mutation is generated, including
direct and indirect mutagenesis.
AGCTTCCTA
TCGAAGGAT
E.g.
1, Direct
mutagenesis
The stable,
unrepaired
base with
altered base
pairing
properties in
the DNA is
fixed to a
mutation
during DNA
replication.
1.Base analog
incorporation
OH
H
Br
:G
O
enol form
H
Br
O
Keto form
5-BrU
AGCTBCCTA
TCGAAGGAT
1st round
of replication
AGCTTCCTA
TCGAAGGAT
AGCTBCCTA
TCGAGGGAT
: A 3. 2nd round
of replication
AGCTBCCTA
TCGAAGGAT
AGCTCCCTA
TCGAGGGAT
A·TG·C transition
2-Indirect mutagenesis
The mutation is introduced as a result of an errorprone repair.
Translesion 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.g. E. coli translesion replication: SOS response
F3 & F4 DNA damage and repair
Physical and chemical Mutagens
minor or
moderate lesions
Error-free
Repairing
Completely repaired
DNA damage
(lesions)
The lesions of extensive,
right ahead of
Replication Fork
Error-prone
repairing
mutations
F2 DNA damage-1
DNA lesion is an alteration to the normal chemical or
physical structure of the DNA.
1, Oxidative damage
(氧化损伤）
1. Occurs under
Normal condition
2. Increased by
ionizing radiation
(physical mutagens)
DNA lesions
(DNA损害）
3，Alkylation
(烷基化作用）
Alkylating agents
(Chemical mutagens)
2, Bulky adducts
(加合物）
UV light
(physical mutagens)
Carcinogen
(Chemical mutagens)
F2
DNA damage-2
The biological effect of the unrepaired DNA lesions
Physical distortion in
the DNA
Blocks replication
And/or transcription
Lethal
(cell death)
The altered chemistry of the bases
may lead to loss of base pairing or
altered base pairing. If such a
lesion was allowed to remain in the
DNA,
Living cell
A mutation could become fixed
by direct or indirect mutagenesis
Mutagenic
F2 DNA damage-3
Spontaneous DNA lesions
Because of inherent chemical reactivity of the DNA and
the presence of normal, reactive chemical species
within the cell
1. Deamination (转氨作用）: CU; methylcytosine T, hard
to be detected
2. Depurination (脱瞟呤作用) : break of the glycosylic bond,
non-coding lesion.
3. Depyrimidine (脱嘧啶作用)
F2 DNA damage-4
Oxidative damage
1- Is a kind of DNA lesions caused by reactive oxygen species
such as superoxide and hydroxyl radicals, which occurs under
normal conditions.
2-The level of this damage can be INCREEASED by hydroxyl
radicals from the radiolysis of water caused by ionizing radiation.
Oxidation products
F2 DNA damage-5
Alkylation
Alkylating agents are electrophilic chemicals which
readily add alkyl (e.g. methyl) groups to various
positions on nucleic acids distinct from those
methylated by normal methylating enzymes such as
methylmethane sulfonate （甲基甲烷磺酸盐）and
ethylnitrosourea (乙基亚硝基脲).
alkylating agents
Alkylated bases
F2 DNA damage-6
Bulky adducts
A kind of 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-1
Photoreactivation
Cyclobutane pyrimidine dimers can be monomerized again
by DNA photolyases (photoreactivating enzymes) in the
presence of visible light.
Direct reversal of a lesion and is error-free
F3
DNA repair-2
Alkyltransferase
Removes the alkyl group from mutagenic O6alkylguanine 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
F3
DNA repair-3
Excision repair
1. There are two forms, 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 either sides
of the lesions (in E.coli
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
1.
2.
3.
modified bases are recognized by
relatively specific DNA
glycosylases which cleave the Nglycosylic bond between the
altered base and the sugar),
leaving an apurinic or
apyrimidinic (AP) site.
An AP endonuclease then cleaves
the DNA at this site and a gap
may be created by further
exonuclease activity. The gap is
generally larger in NER and can
be as small as one nucleotide in
BER.
The gap is filled by DNA
polymerase I and sealed by ligase
F3 DNA repair-4
Mismatch repair
Is a specialized form of excision repair that
deals with any base mispairs produced
during replication and that 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
DNA Repair Mechanisms
The proteins of DNA replication also play roles in the life-preserving repair
mechanisms, helping to ensure the extract replication of template DNA.
F4 Recombination
Includes homologous recombination, site-specific
recombination and transposition.
Recombination is an important reason for variable
DNA sequences among different populations of the
same species.
Homologous recombination
( 同源重组）
Also known as general recombination, this process
involves the exchange of homologous regions
between two DNA molecules.
1- In diploid eukaryotes: commonly occurs during meiosis
by crossing over.
2- In Haploid prokaryotes: recA-dependent, Holliday
model
3- DNA repair in replication fork
Haploid prokaryotes recombination
Between the two homologous DNA duplex
1.
between the replicated portions of a partially
duplicated DNA
2. between the chromosomal DNA and acquired “foreign”
DNA
Holliday model
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’
4. RecA-ssDNA filaments
search the opposite DNA
duplex for corresponding
sequence (invasion).
5. form a four-branched
Holliday structure
6. Branch migration
Resolving Holliday junction
Recombination based DNA
repair at replication fork, also
called post-replication repair
a. Replication encounter a
DNA lesion
b. Skip the lesion & re-initiate
on the other side of the
lesion
c. Fill the daughter strand gap
by replacing it with the
corresponding section from
the parental sister strand by
recombination
d. The original lesion can be
removed later by normal
excision repair.
Site-specific recombination
1. Exchange of non-homologous but specific
pieces of DNA
2. Mediated by proteins that recognize specific
DNA sequences.
bacteriophage λ insertion
1. A λ-encoded integrase (Int): makes staggered cuts in the
specific sites with 7 bp overhangs
2. Int and IHF (integration host factor encoded by bacteria):
promote recombination between these sites and insertion
of the λ DNA into the host chromosome (prophage)
3. λ-encoded excisionase: excision of the phage DNA and the
process reverse
Site-specific recombinases such as FLP, Cre, and C31 have
emerged as powerful tools to manipulate genomes of
eukaryotic model organisms.
Antibody diversity
In eukaryotes, site-specific recombination is responsible for
the generation of antibody diversity.
Immunoglobulins are composed of two heavy (H) chains
and two light (L) chains of various types, both of which
contain regions of constant and variable amino acid
sequence.
The sequences of the variable regions of these chains in
germ cells are encoded by three gene segments: V, D and J.
Antibody diversity
There are a total of 250 V, 15 D and five J genes for H chains
and 250 V and four J for L chains.
Recombination between these segments during differentiation
of antibody-producing cells can produce an enormous number
(> 108) of different H and L gene sequences and hence
antibody specificities.
Transposition
Transposons or transposable elements are small
DNA sequences that can move to virtually any
position in a cell’s genome. Transposition has also
been called illegitimate recombination.
1-it requires no homology between sequences nor is it sitespecific
2-it is relatively inefficient
3-Require Transposase encoded by the transposon ( 转座 子）
Various transposons
In E. coli:
• IS elements/insertion sequence, 1-2 kb in length, comprise
a transposase gene flanked by a short inverted terminal
repeats (ITR, ~20 bp)
• Tn transposon series carry transposition elements and βlactamase (penicillin resistance)
Eukaryotic transposons, many are retrotransposons:
Yeast Ty element encodes protein similar to RT (reverse
transcriptase).
E.g. P-element, piggyBac transposon
Simplified
Transposition
process