UC DAVIS (US)—Two research teams have taken a key step toward understanding the origins of familial breast cancer, opening new possibilities for diagnosing and perhaps treating the disease.
Scientists at the University of California at Davis 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.
BRCA2 is known to be involved in repairing damaged DNA, but exactly how it works with other molecules to repair DNA has been unclear, says Stephen Kowalczykowski, professor of microbiology and senior author of the Nature paper.
“Having the purified protein makes possible far more detailed studies of how it works,” he says.
Kowalczykowski’s group has purified the protein from human cells; another group led by Wolf-Dietrich Heyer, also a professor of microbiology, used genetic engineering techniques to manufacture the human protein in yeast.
The two approaches are complementary, Heyer says, 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 adds.
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 says. Your DNA is constantly suffering damage, even if you avoid exposure to carcinogens. If that damage is not repaired, errors start to accumulate, Kowalczykowski explains. 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 says.
After testing many different cell lines, researchers succeeded in introducing a BRCA2 gene into a human cell line and expressing (producing) it as a whole protein. The team tested the purified protein for its function in repairing damaged DNA.
They found that a much smaller protein called DSS1 stimulated BRCA2 to assemble functional RAD51/DNA complexes. The researchers 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 says.
A more exciting possibility, he adds, 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 says.
The work was supported in part by the National Institutes of Health, the U.S. Department of Defense Breast Cancer Research Program, the Susan G. Komen Breast Cancer Foundation, and American Cancer Society.
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