Download DNA damage, repair and recombination

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MUTAGENESIS
DNA DAMAGE
DNA REPAIR
RECOMBINATION
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Mutations are heritable permanent changes in
the base sequence of DNA.
Point mutations may be transitions (e.g.
GC→AT) or transversions (e.g. GC→TA).
Deletions and insertions involve the loss or
addition of bases and can cause frameshifts in
reading the genetic code. Silent mutations have
no phenotypic effect, while missense and
nonsense mutations change the amino acid
sequence of the encoded protein.
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The high accuracy of DNA replication (one
error per 1010 bases incorporated)
depends on a combination of proper base
pairing of template strand and incoming
nucleotide in the active site of the DNA
polymerase, proofreading of the
incorporated base by 3→5 exonuclease and
mismatch repair.
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Ionizing (e.g. X- and -rays) and nonionizing
(e.g. UV) radiation produce a variety of DNA
lesions.
Pyrimidine dimers are the commonest
product of UV irradiation.
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Base analogs can mispair during DNA
replication to cause mutations.
Nitrous acid deaminates cytosine and
adenine.
Alkylating and arylating agents generate a
variety of adducts that can block
transcription and replication and cause
mutations by direct or, more commonly,
indirect mutagenesis.
Most chemical mutagens are carcinogenic.
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If a base analog or modified base whose base
pairing properties are different from the
parent base is not removed by a DNA repair
mechanism before passage of a replication
fork, then an incorrect base will be
incorporated. A second round of replication
fixes the mutation permanently in the DNA.
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Most lesions in DNA are repaired by errorfree direct reversal or excision repair
mechanisms before passage of a replication
fork. If this is not possible, an error-prone
form of trans- lesion DNA synthesis may take
place involving specialized DNA polymerases
and one or more incorrect bases become
incorporated opposite the lesion.
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The chemical reactivity of DNA with
exogenous chemicals or radiation can give
rise to changes in its chemical or physical
structure. These may block replication or
transcription and so be lethal, or they may
generate mutations through direct or indirect
mutagenesis. The chemical instability of DNA
can generate spontaneous lesions such as
deamination and depurination.
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Reactive oxygen species such as superoxide
and hydroxyl radicals produce a variety of
lesions including 8-oxoguanine and 5formyluracil.
Such damage occurs spontaneously but is
increased by some exogenous agents
including γ-rays.
Alkylation
Electrophilic alkylating agents such
as methylmethane sulfonate and
ethylnitrosourea can modify
nucleotides in a variety of positions.
Most lesions are indirectly
mutagenic, but O6-alkylguanine is
directly mutagenic.
Bulky adducts
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Bulky lesions such as pyrimidine dimers and
arylating agent adducts distort the double
helix and cause localized denaturation. This
disrupts the normal functioning of the DNA.
DNA REPAIR
Photoreactivation
Cleavage of the cyclobutane ring of
pyrimidine dimers by DNA photolyases
restores the original DNA structure.
Photolyases have chromophores which
absorb blue light to provide energy for
the reaction.
Alkyltransferase
An inducible protein specifically
removes an alkyl group from
the O6 position of guanine and
transfers it to itself, causing
inactivation of the protein.
Excision repair
In nucleotide excision repair, an endonuclease makes nicks on either
side of the lesion, which is then removed to leave a gap. This gap is filled
by a DNA polymerase, and DNA ligase makes the final phosphodiester
bond.
In base excision repair, the lesion is removed by a specific DNA
glycosylase.
The resulting AP site is cleaved and expanded to a gap by an AP
endonuclease plus exonuclease. Thereafter, the process is like
nucleotide excision repair.
Mismatch repair
Replication errors which escape proofreading have a
mismatch in the daughter strand.
Hemimethylation of the DNA after replication allows the
daughter strand to be distinguished from the parental
strand.
The mismatched base is removed from the daughter
strand by an excision repair mechanism.
Hereditary
repair defects
Mutations in excision repair genes or a translesion DNA polymerase cause different forms of
xeroderma pigmentosum, a sun-sensitive
cancer-prone disorder.
Excision repair is also defective in Cockayne
syndrome.
Figure 18.8
RECOMBINATION
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Also known as general, this process
involves the exchange of recombination
homologous regions between two DNA
molecules (during meiosis in diploid
eukaryotes)
Haploid bacteria perform recombination, for
example between the replicated portions of a
partially duplicated DNA or between the
chromosomal DNA and acquired ‘foreign’
DNA such as plasmids or phages.
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Homologous recombination is also important for DNA
repair.
When a replication fork encounters an unrepaired,
noncoding lesion it can skip the damaged section of DNA
and re-initiate on the other side, leaving a daughter
strand gap.
This gap can be filled by replacing it with the
corresponding section from the parental sister strand by
recombination.
The resulting gap in the parental sister strand can be filled
easily since it is not opposite a lesion.
The original lesion can be removed later by normal excision
repair. This mechanism has also been called postreplication repair.
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This involves the exchange of nonhomologous
but specific pieces of DNA and is mediated by
proteins that recognize specific DNA
sequences.
It does not require RecA or ssDNA. eg.
Bacteriophage λ
In eukaryotes, site-specific recombination is
responsible for the generation of antibody
diversity.
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Transposons are small DNA sequences that can move to
virtually any position in a cells genome. it requires no
homology between sequences nor is it site-specific.
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the E. coli IS elements or insertion sequences, are 1–2 kb in
length and comprise a transposase gene flanked by short
(~20 bp) inverted terminal repeats (identical sequences
but with opposite orientation). The transposase makes a
staggered cut in the chromosomal DNA and, in a replicative
process, a copy of the transposon inserts at the target site
The gaps are filled and sealed by DNA polymerase and DNA
ligase
The gene into which the
transposon inserts is usually
inactivated, and genes between
two copies of a transposon can
be deleted by recombination
between them.
In addition to a transposase, the Tn transposon series
carry other genes, including one for β -lactamase,
which confers penicillin resistance on the organism
Many eukaryotic transposons have a structure similar to
retroviral genomes
The dispersed repetitive sequences found in higher
eukaryotic DNA (e.g. LINES and SINES) probably
spread through the genome by transposition