Protein made by breast cancer gene purified

A key step in understanding the origins of familial breast cancer hasbeen made by two teams of scientists at the University of California,Davis. The researchers have purified, for the first time, the proteinproduced by the breast cancer susceptibility gene BRCA2 and used itto 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 newpossibilities for understanding, diagnosing and perhaps treatingbreast cancer.

BRCA2 is known to be involved in repairing damaged DNA, but exactlyhow it works with other molecules to repair DNA has been unclear,said Stephen Kowalczykowski, distinguished professor of microbiologyin the UC Davis College of Biological Sciences, UC DavisCancer Center member and senior author of the Nature paper.

"Having the purified protein makes possible far more detailed studiesof 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 UCDavis Department of Microbiology and leader of the Cancer Center'smolecular oncology program, used genetic engineering techniques tomanufacture the human protein in yeast. That work is published inNature Structural and Molecular Biology.

The two approaches are complementary, Heyer said, and the two teamshave been talking and cooperating throughout.

"It's nice to be able to compare the two and see no disagreementsbetween the results," Heyer said.

Experiments with the BRCA2 protein confirm that it plays a role inrepairing damaged DNA. It acts as a mediator, helping anotherprotein, RAD51, to associate with a single strand of DNA andstimulating its activity. One BRCA2 molecule can bind up to sixmolecules of RAD51.

The RAD51/DNA complex then looks for the matching strand of DNA fromthe other chromosome to make an exact repair.

If the BRCA2/RAD51 DNA repair system is not working, the cell resortsto 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 ifyou avoid exposure to carcinogens. If that damage is not repaired,errors start to accumulate, Kowalczykowski said. Those errors caneventually lead to cancer.

The BRCA2 gene was discovered in 1994. Mutations in BRCA2 areassociated with about half of all cases of familial breast andovarian cancer (cases where the propensity to develop cancer seems tobe 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, aftertesting many different cell lines, succeeded in introducing a BRCA2gene into a human cell line and expressing (producing) it as a wholeprotein. Jensen and another postdoc, Aura Carreira, tested thepurified protein for its function in repairing damaged DNA.

Jie Liu, a postdoctoral researcher in Heyer's lab, found that a muchsmaller protein called DSS1 stimulated BRCA2 to assemble functionalRAD51/DNA complexes. Together with Liu, staff research associateTammy Doty and UC Davis undergraduate student Bryan Gibson (now adoctoral student at Cornell University) purified the human BRCA2 andDSS1 proteins from yeast.

One application of the purified protein would be to make antibodiesto BRCA2 that could be used in test kits as a supplement to existinggenetic tests, Kowalczykowski said.

A more exciting possibility, he said, would be to use the system toscreen for drugs that activate or inhibit the interaction betweenBRCA2, RAD51 and DNA. Many cancer treatments work by creating breaksin DNA, and a drug that selectively shuts down a specific DNA repairpathway -- making it harder for cancer cells to recover -- could makethe cells more vulnerable to treatment. That strategy is alreadybeing exploited by a new class of drugs called PARP inhibitors,currently in clinical trials. PARP inhibitors target an alternate DNArepair pathway that cells use when the BRCA2 repair pathway is notavailable.

The BRCA2 protein can also be used to study how different mutationsaffect the gene's function.

"We're just starting to scratch the surface and understand more ofthe mechanisms and interaction with other factors," Kowalczykowskisaid.

Source: University of California - Davis - Health System