Transcript Gene mutation

Biological repair mechanisms
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There are many potential threats to the fidelity of DNA replication.
Not only is there an inherent error rate for the replication of DNA, but
there are also spontaneous lesions that can provoke additional errors.
Moreover, mutagens in the environment can damage DNA and greatly
increase the mutation rate.
Living cells have evolved a series of enzymatic systems that repair
DNA damage in a variety of ways. Failure of these systems can lead
to a higher mutation rate. A number of human diseases including
certain types of cancer can be attributed to defects in DNA repair, as
we shall see later. Let's first examine some of the characterized repair
pathways and then consider how the cell integrates these systems into
an overall strategy for repair.
We can divide repair pathways into several categories.
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Prevention of errors before they happen
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Some enzymatic systems neutralize potentially damaging compounds
before they even react with DNA. One example of such a system is
the detoxification of superoxide radicals produced during oxidative
damage to DNA: the enzyme superoxide dismutase catalyzes the
conversion of the superoxide radicals into hydrogen peroxide, and the
enzyme catalase, in turn, converts the hydrogen peroxide into water.
Another error-prevention pathway depends on the protein product of
the mutT gene: this enzyme prevents the incorporation of 8-oxodG,
which arises by oxidation of dGTP, into DNA by hydrolyzing the
triphosphate of 8-oxodG back to the monophosphate
DNA damage products formed after attack by
oxygen radicals. dR = deoxyribose
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Direct reversal of damage
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The most straightforward way to repair a lesion, once it occurs, is to reverse it
directly, thereby regenerating the normal base. Reversal is not always possible,
because some types of damage are essentially irreversible. In a few cases, however,
lesions can be repaired in this way. One case is a mutagenic photodimer caused by
UV light. The cyclobutane pyrimidine photodimer can be repaired by a photolyase
that has been found in bacteria and lower eukaryotes but not in humans. The enzyme
binds to the photodimer and splits it, in the presence of certain wavelengths of
visible light, to generate the original bases. This enzyme cannot operate in the dark,
so other repair pathways are required to remove UV damage. A photolyase that
reverses the 6-4 photoproducts has been detected in plants and Drosophila.
Repair of a UV-induced pyrimidine photodimer by a
photoreactivating enzyme, or photolyase. The enzyme
recognizes the photodimer (here, a thymine dimer) and
binds to it. When light is present, the photolyase uses
its energy to split the dimer into the original monomers.
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Removal of alkyl groups
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Alkyltransferases also are enzymes taking part in the direct reversal of lesions.
They remove certain alkyl groups that have been added to the O-6 positions of
guanine by such agents as NG and EMS. The methyltransferase from E. coli has
been well studied. This enzyme transfers the methyl group from O-6-methylguanine
to a cysteine residue on the protein. When this happens, the enzyme is inactivated,
so this repair system can be saturated if the level of alkylation is high enough.
Alkylation-induced specific mispairing. The
alkylation (in this case, EMS-generated ethylation)
of the O-6 position of guanine and the O-4 position
of thymine can lead to direct mispairing with
thymine and guanine, respectively, as shown here.
In bacteria, where mutations have been analyzed in
great detail, the principal mutations detected are
GC → AT transitions, indicating that the O-6
alkylation of guanine is most relevant to
mutagenesis.
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Excision-repair pathways
Also termed nucleotide excision
repair, this system includes the
breaking of a phosphodiester
bond on either side of the lesion,
on the same strand, resulting in
the excision of an
oligonucleotide. This excision
leaves a gap that is filled by
repair synthesis, and a ligase
seals the breaks. In prokaryotes,
12 or 13 nucleotides are
removed; whereas, in
eukaryotes, from 27 to 29
nucleotides are eliminated.
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The excinuclease
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In E. coli, the products of the uvrA, B, and C genes constitute the
excinuclease. The UvrA protein, which recognizes the damaged DNA,
forms a complex with UvrB and leads the UvrB subunit to the damage
site before dissociating. The UvrC protein then binds to UvrB. Each of
these subunits makes an incision. The short DNA 12-mer is unwound
and released by another protein, helicase II.
The human excinuclease is considerably
more complex than its bacterial counterpart
and includes at least 17 proteins. However,
the basic steps are the same as those in E.
coli
Schematic representation of events following incision
by UvrABC exinuclease in E. coli.
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Excision repair defects in humans
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Several human genetic diseases are known to be due to repair defects.
Xeroderma pigmentosum (XP) results from a defect in any of the
genes (complementation groups) effecting nucleotide excision repair.
People suffering from this disorder are extremely prone to UVinduced
skin cancers as a result of exposure to sunlight and have frequent
neurological abnormalities.
Nucleotide excision repair is coupled to transcription. This model for coupled repair in
mammalian cells shows RNA polymerase (pink) pausing when encountering a lesion. It
undergoes a conformational change, allowing the DNA strands at the lesion site to
reanneal.
Protein factors aid in coupling
by bringing TFIIH and other
factors to the site to carry out
the incision, excision, and repair
reactions. Then transcription can
continue normally.
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Specific excision pathways
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Certain lesions are too subtle to cause a distortion
large enough to be recognized by the uvrABCencoded general excision-repair system and its
counterparts in higher cells. Thus, additional
excision pathways are necessary.
DNA glycosylase repair pathway (base-excision
repair). DNA glycosylases do not cleave
phosphodiester bonds, but instead cleave Nglycosidic (base–sugar) bonds, liberating the
altered base and generating an apurinic or an
apyrimidinic site, both called AP sites, because
they are biochemically equivalent. The resulting
Ap site is then repaired by an AP endonuclease
repair pathway
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Mismatch repair
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Some repair pathways are capable of recognizing errors even after
DNA replication has already occurred. One such system, termed the
mismatch repair system, can detect mismatches that occur in DNA
replication. Suppose you were to design an enzyme system that could
repair replication errors. What would this system have to be able to
do? At least three things:
1. Recognize mismatched base pairs.
2. Determine which base in the mismatch is the incorrect one.
3. Excise the incorrect base and carry out repair synthesis.
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The second point is the crucial property of such a system. Unless it is
capable of discriminating between the correct and the incorrect bases,
the mismatch repair system could not determine which base to excise.
If, for example, a G–T mismatch occurs as a replication error, how can
the system determine whether G or T is incorrect? Both are normal
bases in DNA. But replication errors produce mismatches on the
newly synthesized strand, so it is the base on this strand that must be
recognized and excised.
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DNA methylation
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To distinguish the old, template strand from the newly synthesized
strand, the mismatch repair system in bacteria takes advantage of the
normal delay in the postreplication methylation of the sequence
The methylating enzyme is adenine methylase, which creates 6methyladenine on each strand. However, it takes the adenine
methylase several minutes to recognize and modify the newly
synthesized GATC stretches. During that interval, the mismatch repair
system can operate because it can now distinguish the old strand from
the new one by the methylation pattern. Methylating the 6-position of
adenine does not affect base pairing, and it provides a convenient tag
that can be detected by other enzyme systems.
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Mismatch repair
Model for mismatch repair in E. coli.
Because DNA is methylated by
enzymatic reactions that recognize the A
in a GATC sequence, the newly
synthesized strand will not be methylated
directly after DNA replication.
The hemimethylated DNA duplex serves
as a recognition point for the mismatch
repair system in discerning the old from
the new strand. Here a G–T mismatch is
shown. The mismatch repair system can
recognize and bind to this mismatch,
determine the correct (old) strand
because it is the methylated strand of a
hemimethylated duplex, and then excise
the mismatched base from the new
strand. Repair synthesis restores the
normal base pair.
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The complex MutS-MutH
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Steps in E. coli mismatch repair.
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(1) MutS binds to mispair.
(2) MutH and MutL are recruited to form a
complex. MutH cuts the newly synthesized
(unmethylated) strand, and exonuclease
degradation goes past the point of the mismatch,
leaving a patch.
(3) Single-strand-binding protein (Ssb) protects
the single-stranded region across from the
missing patch.
(4) Repair synthesis and ligation fill in the gap.
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Mismatch repair defects in humans
Hereditary nonpolyposis colorectal cancer (HNPCC) is one
of the most common inherited predispositions to cancer,
affecting as many as 1 in 500 people in the Western world.
Most HNPCC results from a defect in genes that encode the
human counterparts (and homologs) of the bacterial MutS and
MutL proteins. The inheritance of HNPCC is autosomal
dominant. Cells with one functional copy of the mismatch
repair genes have normal mismatch repair activity, but tumor
cell lines arise from cells that have lost the one functional
copy and are thus mismatch repair deficient. These cells
display high mutation rates that eventually result in tumor
growth and proliferation.
Mismatch repair in humans. (1) Mispairs and misaligned bases
arise in the course of replication. (2) The G–T-binding protein
(GTBP) and the human MutS homolog (hMSH2) recognize the
incorrect matches. (3) Two additional proteins, hPMS2 and
hMLH1, are recruited and form a larger repair complex. (4) The
mismatch is repaired after removal, DNA synthesis, and ligation.
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SOS repair
DNA damage often results ina replication block, because DNA synthesis will not
proceed past a base that cannot specify its complementary partner by hydrogen
bonding. In bacterial cells, such replication blocks can be bypassed by inserting
nonspecific bases. The process requires the activation of a special system, the SOS
system. The name SOS comes from the idea that this system is induced as an
emergency response to prevent cell death in the presence of significant DNA damage.
SOS induction is a last resort, allowing the cell to trade death for a certain level of
mutagenesis.
The recA gene, takes part in postreplication
repair. Here the DNA replication system stalls at
a UV photodimer and then restarts past the
block, leaving a single-stranded gap. This
process leads to few errors.
SOS bypass, in contrast, is highly mutagenic.
Here the replication system continues past the
lesion, accepting noncomplementary
nucleotides for new strand synthesis
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Schemes for postreplication repair
(a) In recombinational repair, replication jumps across a blocking lesion, leaving a gap
in the new strand. A recA-directed protein then fills the gap, using a piece from the
opposite parental strand (because of DNA complemen-tarity, this filler will supply the
correct bases for the gap). Finally, the RecA protein repairs the gap in the parental
strand.
(b) In SOS bypass, when
replication reaches a blocking
lesion, the SOS system inserts
the necessary number of bases
(often incorrect ones) directly
across from the lesion and
replication continues without a
gap. Note that with either
pathway the original blocking
lesion is still there and must be
repaired by some other repair
pathway.
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Summary of repair mechanisms
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We can now assess the overall repair system strategy used by the cell. It
would be convenient if enzymes could be used to directly reverse each
specific lesion. However, sometimes that is not chemically possible, and not
every possible type of DNA damage can be anticipated.
Therefore, a general excision repair system is used to remove any type of
damaged base that causes a recognizable distortion in the double helix.
When lesions are too subtle to cause such a distortion, specific excision
systems, glycosylases, or removal systems are designed.
To eliminate replication errors, a postreplication mismatch repair system
operates; finally, postreplication recombinational systems eliminate gaps
across from blocking lesions that have escaped the other repair systems.
The SOS system is the last resort for a cell to survive a potentially lethald
DNA damage
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DNA repair and mutation rates
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The repair processes are so efficient that the observed base substitution rate is as low
as 10−10 to 10−9 per base pair per cell per generation in E. coli. However, mutant
strains with increased spontaneous mutation rates have been detected. Such strains
are termed mutators. In many cases, the mutator phenotype is due to a defective
repair system. In humans, these repair defects often lead to serious diseases.
In E. coli, the mutator loci mutH, mutL, mutU, and mutS affect components of the
postreplication mismatch repair system, as does the dam locus, which specifies the
enzyme deoxyadenosine methylase. Strains that are Dam− cannot methylate
adenines at GATC sequences, and so the mismatch repair system can no longer
discriminate between the template and the newly synthesized strands. This failure to
discriminate leads to a higher spontaneous mutation rate.
Mutations in the mutY locus result in GC → TA transversions, because many G–A
mispairs and all 8-oxodG–A mispairs are unrepaired. The mutM gene encodes a
glycosylase that removes 8-oxodG. Strains lacking mutM are mutators for the GC →
TA transversion. Strains that are MutT− have elevated rates of the AT → CG
transversion, because they lack an activity that prevents the incorporation of 8oxodG across from adenine.
Strains that are Ung− are missing the enzyme uracil DNA glycosylase. These
mutants cannot excise the uracil resulting from cytosine deaminations and, as a
result, have elevated levels of C → T transitions. The mutD locus is responsible for a
very high rate of mutagenesis (at least three orders of magnitude higher than
normal). Mutations at this locus affect the proofreading functions of DNA
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