Protein made by breast cancer gene purified
A key step in understanding the origins of familial breast cancer has
been made by two teams of scientists at the University of California,
Davis. The researchers have purified, for the first time, the protein
produced by the breast cancer susceptibility gene BRCA2 and used it
to study the oncogene’s role in DNA repair.
The results will be published online Aug. 22 in the journals Nature,
and Nature Structural and Molecular Biology. They open new
possibilities for understanding, diagnosing and perhaps treating
breast cancer.
BRCA2 is known to be involved in repairing damaged DNA, but exactly
how it works with other molecules to repair DNA has been unclear,
said Stephen Kowalczykowski, distinguished professor of microbiology
in the UC Davis College of Biological Sciences, UC Davis
Cancer Center member and senior author of the Nature paper.
“Having the purified protein makes possible far more detailed studies
of how it works,” Kowalczykowski said.
Kowalczykowski’s group has purified the protein from human cells;
another group led by Professor Wolf-Dietrich Heyer, also in the UC
Davis Department of Microbiology and leader of the Cancer Center’s
molecular oncology program, used genetic engineering techniques to
manufacture the human protein in yeast. That work is published in
Nature Structural and Molecular Biology.
The two approaches are complementary, Heyer said, and the two teams
have been talking and cooperating throughout.
“It’s nice to be able to compare the two and see no disagreements
between the results,” Heyer said.
Experiments with the BRCA2 protein confirm that it plays a role in
repairing damaged DNA. It acts as a mediator, helping another
protein, RAD51, to associate with a single strand of DNA and
stimulating its activity. One BRCA2 molecule can bind up to six
molecules of RAD51.
The RAD51/DNA complex then looks for the matching strand of DNA from
the other chromosome to make an exact repair.
If the BRCA2/RAD51 DNA repair system is not working, the cell resorts
to other, more error-prone methods.
“It’s at the apex of the regulatory scheme of DNA repair,”
Kowalczykowski said. Your DNA is constantly suffering damage, even if
you avoid exposure to carcinogens. If that damage is not repaired,
errors start to accumulate, Kowalczykowski said. Those errors can
eventually lead to cancer.
The BRCA2 gene was discovered in 1994. Mutations in BRCA2 are
associated with about half of all cases of familial breast and
ovarian cancer (cases where the propensity to develop cancer seems to
be hereditary), and are the basis for genetic tests.
But purifying the protein made by the gene has proved difficult.
“It’s very large, it does not express well, and it degrades easily,”
Kowalczykowski said.
Ryan Jensen, a postdoctoral researcher in Kowalczykowski’s lab, after
testing many different cell lines, succeeded in introducing a BRCA2
gene into a human cell line and expressing (producing) it as a whole
protein. Jensen and another postdoc, Aura Carreira, tested the
purified protein for its function in repairing damaged DNA.
Jie Liu, a postdoctoral researcher in Heyer’s lab, found that a much
smaller protein called DSS1 stimulated BRCA2 to assemble functional
RAD51/DNA complexes. Together with Liu, staff research associate
Tammy Doty and UC Davis undergraduate student Bryan Gibson (now a
doctoral student at Cornell University) purified the human BRCA2 and
DSS1 proteins from yeast.
One application of the purified protein would be to make antibodies
to BRCA2 that could be used in test kits as a supplement to existing
genetic tests, Kowalczykowski said.
A more exciting possibility, he said, would be to use the system to
screen for drugs that activate or inhibit the interaction between
BRCA2, RAD51 and DNA. Many cancer treatments work by creating breaks
in DNA, and a drug that selectively shuts down a specific DNA repair
pathway — making it harder for cancer cells to recover — could make
the cells more vulnerable to treatment. That strategy is already
being exploited by a new class of drugs called PARP inhibitors,
currently in clinical trials. PARP inhibitors target an alternate DNA
repair pathway that cells use when the BRCA2 repair pathway is not
available.
The BRCA2 protein can also be used to study how different mutations
affect the gene’s function.
“We’re just starting to scratch the surface and understand more of
the mechanisms and interaction with other factors,” Kowalczykowski
said.
