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SPARQ-ed Immersion: Mutations in the
Tumour Suppressor Gene p53
Cancer and the Cell Cycle
The cell cycle represents the normal progression of cells through the division cycle to produce two identical
daughter cells. When a cell is given the signal to divide, normal function is suspended and all of the cell’s
resources are directed to replication of DNA and duplication of organelles. When this occurs, the cells cannot
carry out their normal role until division is complete. As a result, most cells do not continuously divide, as
this would interfere with normal body function.
Progression through the cell cycle is controlled by a series of checkpoints. If a cell cannot meet the conditions
needed at each checkpoint, the cycle arrests at that point. This prevents cells being duplicated with significant
errors. If something occurs which interferes with the regulation of the cell cycle, cells may enter into a state
of continuous division. This not only increases the number of cells present, the cells that are formed cannot
carry out their normal function. This hyperproliferation is one of the hallmarks of the range of conditions
called cancer. A deeper understanding of the mechanisms of checkpoint regulation can help us understand
the factors which lead to the development and progression of cancer, as well as indicating potential targets
for chemotherapy.
p53 as a Tumour Suppressor
The progression of cells through the cell cycle is governed by a complex array of interlinked proteins.
p53 is a protein involved in the regulation of the cell cycle. If the DNA is damaged by radiation (such as UV
radiation from sunlight) or a chemical agent, the protein ATM (Ataxia Telangiectasia Mutated) is expressed.
One of ATM’s roles is to attach phosphate groups to p53, creating an active form which is protected from
degradation by the cell’s normal processes. This allows levels of p53 to accumulate in the cell. Once the levels
of p53 reach a critical level, it binds to the DNA and activates the expression of the protein p21 which causes
cell cycle arrest by inhibiting the activity of CDK-cyclin complexes. This pauses progression through the cell
cycle until the DNA repair mechanisms in the cell can fix the problem and prevent the cell from entering into
mitosis with significant DNA damage or mutations.
In addition, if the damage to DNA is so severe that it cannot be repaired, p53 will bind to the DNA and activate
key genes (eg. PUMA) which trigger apoptosis (programmed cell death). In this way, in the presence of
dangerous damage or mutations to the DNA, p53 acts both as a “brake” on progression through the cell cycle
and cell division, and as a promoter of the removal of cells with damaged DNA through apoptosis (Figure 2).
As a result, p53 plays an important role in preventing the hyperproliferation which is a hallmark of cancer. It
is not surprising therefore that mutations in the p53 gene which interfere with its normal function are among
the most common in cancer and are associated with a wide range of cancers.
Mutations in p53
A mutation is any change to the sequence of nucleotides in a DNA strand. The order of nucleotides in a gene
determines the sequence of amino acids in the protein encoded by that gene, so any change in the
nucleotides may change the amino acids which make up the protein. Since it is the amino acids which
determine the secondary and tertiary structure of the protein, a simple change to one or two nucleotides in
the gene may have significant consequences for the function of that protein in the cell.
Mutations to p53 are associated with an increased risk of a wide range of cancers, including colon, breast,
head and neck, lung and leukaemia. p53 performs its regulatory role by binding to the DNA upstream of
genes which encode proteins which arrest the cell cycle. Once bound, p53 activates transcription factors
which transcribe the target genes to mRNA and this leads to the expression of the proteins. If p53 can not
bind to the DNA, these transcription factors will not induce the expression of proteins like p21 and cell
division will be allowed to progress unchecked.
Residue 248 is just one location in the p53 protein in which mutations have been identified which correlate
with an increased risk of cancer. In this project, you will investigate the effects of mutations within the DNA
binding domain of p53 on the expression of protein in a model system, comparing it to the effects of
mutations in another region of the protein.
Project overview:
In this project, participants will use site-directed mutagenesis to create a point mutation in a region of the
p53 gene that encodes an important region of the DNA binding domain, alongside a second mutation in a
region of the gene outside the DNA binding domain. Following a DpnI restriction digest to remove the original
template vector, participants will use these vectors containing the mutant p53 genes to transform E. coli.
After growing the transformed cells on selective medium, they will select colonies to subculture into selective
broth, and, after allowing these subcultures to grow, use a mini-prep procedure to recover sufficient
quantities of the plasmids containing the mutant p53 genes. These plasmids will then be used to transfect
mammalian cells, alongside a plasmid in which a p53 DNA-binding sequence regulates the expression the
luciferase gene and a vector containing the gene for green fluorescent protein (GFP). After ascertaining the
transfection efficiency using fluorescence microscopy (observing the level of cells expressing GFP), the cells
will be lysed and a luciferase assay performed to determine the effects of the mutations on the function of
the p53 gene.
Experimental protocols include:
 Site-directed mutagenesis
 Agarose gel electrophoresis
 Restriction digest
 Bacterial transformation
 Plasmid DNA extraction
 Transfection of eukaryotic cells
 Luciferase assay as a Biochemical marker