Genetic Changes Induced by DNA Interstrand Crosslinks
Early in her presentation, Dr. Saffran introduced us to the applications of DNA
interstrand crosslink (ICL). Dr. Saffran stated that her laboratory studies the repair of
double strand damage inflicted on the DNA of the eukaryotic cells of baker’s yeast
(Saccharomyces cerevisiae) by interstrand crosslinkage induced by the photoreaction of
psoralen. Psoralen, a derivative of furanocoumarin, is used in cancer chemotherapy and
in the treatment of psoriasis. It is a three ringed molecule that intercalates between the
base pairs of the DNA double helix. Under UV radiation, psoralen forms a covalent bond
with a pyrimidine base on each strand of the double helix forming an interstrand
crosslink. Interstrand crosslinks are difficult to repair and therefore are lethal to cells
because they block important processes such as DNA replication and transcription.
However, it is this cytotoxic effect that forms the mechanistic basis of action of many
drugs in cancer therapy. Since cancer is characterized by an uncontrolled cell division,
crosslinking agents are a special topic among scientists. DNA crosslinking agents such
as cisplatin, nitrogen mustards and nitrosourceas are used in the treatment of cancer. In
PUVA therapy (Psoralen +UVA light) patients take psoralen either by ingestion or
topical application and then expose their skin to UVA light. According to Dr. Saffran, in
studies of psoriasis patients treated with PUVA therapy, it was found that there is a dosedependent increase in squamous cell carcinomas, showing that psoralen is a human
carcinogen.
Dr. Saffran emphasized that cancer is a genetic disease. Changes in genes which r
egulate cell growth can lead to the generation of tumor cells known as transformed cells.
1
Dr. Saffran stated that there are two types of altered genes found in tumor cells. First,
there is the oncogenes which when activated can lead to unregulated cell replication.
Proto-oncogenes stimulate cellular replication. They are highly regulated to prevent
excessive cellular proliferation.
Proto-oncogenes are activated to oncogenes by
amplification (extra copies produce more activities), point mutation and translocation to
active promoter. Examples of Proto-oncogenes are growth factors and growth factor
receptors.
Then there are the tumor suppressor genes which when inactivated or lost can lead to
unregulated cell replication.
The function of tumor suppressor gene is to prevent
uncontrolled cell replication.
If they become inactivated through mutation, then
unregulated cell growth can occur leading to a tumor. However, in diploid cells there are
two copies of each tumor suppressor cell. If one of the tumor suppressor cells becomes
inactivated then the other can still carry out the normal function and cell growth is still
regulated. However if the second functional copy is lost, the tumor suppressor will
become inactivated. That is to say that there is a mutation in one copy and loss of
heterozygosity in the other copy.
Dr. Saffran said that all cells contain repair systems which recognize and repair
DNA damage. Crosslinks are repaired by cells but the repair process can induce genetic
changes such as mutations and rearrangements. Dr Saffran stated that in order for the cell
to survive it must find a way to rid itself of the crosslink and continue with the processes
of replication and transcription. Dr. Saffran stated that in Saccharomyces cerevisiae, the
repair of psoralen induced crosslinks can be mutagenic and error- free. However, there
are three DNA repair pathways, Homologous Recombination (HR), Nucleotide Excision
2
Repair (NER) and Post-replication Repair (PRR). The error-free pathway requires
nucleotide excision repair (NER) followed by homologous recombination (HR).
Nucleotide excision repair makes incisions; which are processed to double strand breaks
during DNA replication.
Dr. Saffran stated that homologous recombination involves
genetic exchanges between two DNA molecules or DNA regions on the same molecule
that share a region of nearly identical sequences.
Thus homologous recombination
allows an undamaged homologous gene to act as a template for replacement of the
damaged DNA. Homologous Recombination is a damage repair mechanism following
excision of the potentially lethal interstrand crosslink.
Double strand breaks are
predominantly repaired by homologous recombination. This results in gene conversion,
which involves the genetic copying of information from the undamaged DNA to the
damaged DNA.
Homologous Recombination also results in DNA rearrangement.
Another type of homologous recombination repair is single strand annealing which
results in deletion. The Post-replication repair is an alternative mutagenic pathway. It
allows DNA polymerase to replicate past the damaged DNA and continue with DNA
synthesis. There are two pathways in the post-replication repair pathway, an error prone
branch and an error-free branch which seems to do the same as the homologous
recombination pathway. Dr. Saffran summarized the three repair pathways by saying that
psoralen crosslink repair depends on all three repair systems, that is, nucleotide excision
repair and homologous recombination act together in error-free repair while postreplication repair acts in error-prone repair.
Dr. Saffran then discussed the experiments conducted in her laboratory. Dr.
Saffran stated that the experiment is set up as a direct repeat assay, which consists of two
3
nonfunctional his3 genes with mutations at different sites flanking a functional TRP1
gene as shown in Figure1.
his3-622x
TRP1
his3-207x
Figure 1:Two directly repeated his3 genes flanking a functional TRP 1 gene. XbaI linker
insertions are shown at 207x and 622x (Inside arrangement)
The direct repeat assay measures the loss of heterozygosity by recombination. The
functional HIS3 gene is required for histidine synthesis. Cells lacking a functional HIS3
gene require histidine in the growth medium. Homologous Recombination can generate
a functional copy. Cells with active HIS3 will grow in the absence of added histidine.
Dr. Saffran stated that there are two arrangements which detect different types of
recombination. There is the inside arrangement as shown in Figure 1 and the outside
arrangement as shown in Figure 2.
his3-207x
TRP1
his3-622x
Figure 2 : Two directly repeated his3 genes flanking a functional TRP 1 gene. XbaI
3
linker insertions are shown at 207x and 622x (Outside arrangement)
functional TRP 1 gene. XbaI linker insertions are shown at 207x and 622x (Inside
arrangement)
4
The inside arrangement produces gene conversion as well as single strand
annealing(deletion). After DNA replication but before cell division there are two sister
chromatids. If the two sister chromatids align imperfectly, that is, the damaged sister
chromatid and the undamaged sister chromatid then by gene conversion a functional
HIS3 gene will be produced. The phenotype for these colonies will be His+Trp+.
his3
TRP1
his3
Gene conversion
Or
TRP1
HIS3
his3
his3
His+Trp+ Phenotype
TRP1
HIS3
His+Trp+ Phenotype
Figure 3: Gene Conversion
The inside arrangement can produce His+ recombinants colonies that lack the TRP1 gene,
these colonies have the phenotype His+Trp- and cannot grow without tryptophan.
TRP1
his3
his3
Deletion
HIS 3
His+ Trp- Phenotype
Figure 4: Single Strand Annealing(deletion)
5
The outside arrangement however, produce His+ recombinants by crossing over, this is
known as triplication or unequal sister chromatid exchange.
his3-207X
TRP1
Unequal sister
chromatid exchange
his3-207X
TRP1
TRP1
HIS3
TRP1
his3-622X
his3-622X
Figure 5: Unequal Sister Chromatid Exchange
Dr. Saffran stated that as the concentration of psoralen increases there is a large increase
in the number of His+ colonies per 1000 survivors for the inside arrangement as opposed
to the outside arrangement.
The 950 base pair (bp) sequence that contains a HIS3 gene is amplified by
polymerase chain reaction (PCR). The thermal cycler is set to produce billions of copies
of this particular DNA sequence. Following PCR, the products are subjected to digest by
the restrictive endonuclease XbaI. The XbaI enzyme recognizes the linker insertions in
each of the his3 mutated genes. Incision at the recognition site produces two fragments,
one larger than the other. The 950 bp fragment containing the HIS3 gene does not
contain the recognition site and thus remains uncut.
The technique agarose gel
electrophoresis is used to determine the base pair number of each band in each lane of the
gel. A DNA ladder is used to determine the size of the different fragments. The size of
the bands in XbaI digestion results is used to determine which type of repair mechanism
had occurred in the cell. Gene conversion only converts one of the two non-functional
6
his3 genes into a functional HIS3 gene
Thus, the gel electrophoresis will show a lane
containing a 950 bp fragment plus either an 800 bp fragment or a 600 bp fragment.
Figure 3 shows these two different outcomes in gene conversion. A lane containing only
a band of 950 bp indicates that DNA repair occurred by deletion The TRP1 gene between
the his3 repeats is deleted leaving a single copy of the HIS3 gene as can be seen in Figure
4. A lane which contains bands of 950,800 and 600 indicates that DNA repair occurred
by way of the unequal sister chromatid exchange pathway.
Dr. Saffran mentioned that the major pathway of interstrand crosslink induced
recombination is gene conversion. Deletions and triplications occur at lower frequencies.
However spontaneous recombination has a different distribution: there are more deletions
and triplications and fewer gene conversions. Dr. Saffran said that most interstrand
crosslink induced recombination is error free. Noncrossover recombination between
properly aligned sister chromatids will not produce genetic changes, as a matter of fact
this will not be detected in the assay. Interstrand crosslink induces gene conversion,
deletions and triplications.
These are genetic changes that can lead to loss of
heterozygosity.
Dr. Saffran stated that survival experiments were carried out using different
concentrations of psoralen plus near UV light. Psoralen induced interstrand crosslink
depends on all three of the repair systems. Dr Saffran said that the nucleotide excision
repair and homologous repair pathways act together in error-free repair while the postreplication repair pathway acts in the error-prone repair. Yeast strains which are deficient
in the post-replication repair pathway are extremely sensitive to psoralen induced
interstrand crosslink.
In yeast strains which are deficient in the homologous
7
recombination repair pathways, interstrand crosslink induced recombination is decreased.
Interstrand crosslink induced deletions and triplications in homologous recombinationdeficient strains such as rad51 and rad55.
However in the strains containing the
homologous recombination repair pathway, interstrand crosslink repair induced mostly
deletion. In strains such as rad1 which lacks the nucleotide excision repair pathway,
interstrand, crosslink induced recombination is reduced. The active RAD1 gene in repair
proficient cells is required for the single strand annealing pathway of homologous
recombination which leads to deletions, as well as for nucleotide excision repair.
Interstrand crosslink damage induced in repair deficient strains such a rad1 and rad3 tend
to lead to DNA repair by way of deletion. An increase in unequal sister chromatid
exchange was also observed in rad3 strains. Dr. Saffran stated that recombination is
increased in rad18 post-replication repair-deficient cells. Dr. Saffran concluded her
presentation by saying that when there are deficiencies in the nucleotide excision repair,
homologous recombination and post-replication repair pathways then there will be an
increase in error- prone repair which will result in rearrangement, deletions and
mutations.
8