Part F: A Closer Look at....
Repair of DNA
Organisms have evolved at least four processes for repairing UV damage in
DNA: photoreactivation, excision, error-prone, and recombination repair.
Depending on the type of organism and the nature of the UV damage, these
processes may successfully repair damage, partially repair the damage and create a
mutation, or fail to work at all.
The simplest process for repair of pyrimidine dimers is called
photoreactivation which, as the name suggests, requires light. Photoreactivation is
catalyzed by a single protein called photolyase, which uses the energy in a photon
of light to chemically break apart a pyrimidine dimer in DNA. Photoreactivation
probably represents the earliest type of UV repair system because many species,
from bacteria through marsupials share the enzyme responsible. Humans and
other placental mammals do not seem to have a photoreactivation process, but the
gene which codes for photolyase has been conserved and may have evolved to
play a role in the excision repair process. The PHR1 gene encoding photolyase is
defective in the sensitive strain used in the experiments.
In excision repair, the region of DNA containing the dimer or other damage
is physically cut out and then replaced by new DNA synthesis (Figure 1).
Excision repair has more steps and requires more enzymes than photoreactivation,
but it can work on damage created by agents other than UV and on lesions other
than pyrimidine dimers. In Escherichia coli bacteria, excision repair requires six
proteins: three are involved in finding the damaged region of the DNA and
cutting the DNA strand around the lesion; one participates in removing the
damaged bit; DNA polymerase replaces the portion which was removed; and a
final enzyme called DNA ligase glues the new and old portions back together.
Mutations in the genes coding for any of these proteins will interfere with the
process and cause the mutant bacterium to be highly sensitive to killing and
mutation by UV light. The excision repair system probably repairs a large amount
of UV damage.
In yeast and other eukaryotes, DNA is wrapped up in more complicated
structures than in bacteria, which may explain why these organisms seem to need
more proteins to carry out excision repair. In yeast, at least twelve proteins may
participate in excision repair. Researchers originally identified many of these by
finding mutants unable to repair UV damage. We don't yet know the functions of
all of these proteins, but scientists very recently found that the RAD1 and RAD10
gene products may act together in cutting DNA near dimers and that the RAD3
gene product is needed to identify dimers to the other repair proteins. These genes
have close counterparts in humans: for example, the protein made by the RAD3
gene has the same sequence of amino acids in over 50% of the positions as the
product of its human counterpart, ERCC2. People with mutations in ERCC2 are
very sensitive to sunlight and suffer from the disease xeroderma pigmentosum.
Yeast with mutations in RAD3 are very sensitive to UV and are killed or mutated
by very low doses of UV. RAD1 is mutated in the sensitive strain
The excision process described in the previous section is mostly accurate, or
error-free. Sometimes, however, mistakes are made when a cell tries to repair a
lesion in its DNA. In the case of pyrimidine dimers, mistakes may happen when
two dimers are near each other on opposite strands of the DNA (Figure 2). If the
cell tries to do excision repair, it won't know how to copy the dimer when it tries
to carry out the repair DNA synthesis because the dimer is not a normal part of
DNA. It might make a mistake rather than not repair the gap in the DNA.
Sometimes, unfortunately, an error-prone process is the only way to repair DNA
damage. Most mutations arising after UV treatment of cells are the result of error-prone repair of the DNA lesions. In yeast, we know of several genes whose
products are required for error-prone repair; one of them, RAD18, is mutated in the
sensitive strain used in the experiments.
When pyrimidine dimers block DNA replication in a eukaryotic
chromosome, the polymerase can start replication at other places further
downstream. The result of replicating a DNA molecule or chromosome
containing a dimer is thus a gap in one strand of the DNA where the dimer
blocked a portion from being copied (Figure 3). A gap in DNA means that one
strand is missing information; the strand must be repaired before the cell divides.
The most frequent way that cells fill such a gap is by genetic recombination with
another DNA molecule or chromosome containing the same or similar
information. The recombinational repair system is a fourth process involved in
repair of UV damage to DNA. The genes which make the proteins functioning in
this system have been identified because mutations in them block recombination.
One important member of this group is the RAD51 gene, which makes a protein
that can help DNA molecules find their similar partners and begin recombination.
Figure 1: Steps in Excision Repair
Figure 2: Steps in Error-prone Repair
Figure 3: Steps in Recombinational Repair
Step 1: DNA with dimer is replicated, leaving a gap in one daughter
Step 2: Recombination with other daughter molecule fills gap by transfer of
Step 3: DNA replication fills gap in donor daughter molecule.
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