New insights into a gene most associated with breast cancer, gained with the help of Argonne’s Advanced Photon Source, could explain how DNA is repaired following cell division.

A team of researchers has uncovered new information about a protein that may have major implications for cancer and infertility studies.

The protein, best known for its association with many cases of breast cancer, is guided by the BRCA2 gene, and is critical to repairing breaks in DNA, the molecule that carries genetic information. The breakdown of this repair is a hallmark of many cancers. The research team, led by the University of Michigan, determined the structure of a complex of two proteins — BRCA2 together with MEILB2 — that allows repairs to happen efficiently in cells that are splitting apart.

The team’s results were reported in Nature Structural and Molecular Biology. As part of their research, the team used the resources of the Advanced Photon Source (APS), a U.S. Department of Energy (DOE) Office of Science user facility at DOE’s Argonne National Laboratory.

In germ cells — the cells that give rise to sperm or eggs — DNA breaks occur before the cells undergo cell splitting, or meiosis. The breaks ensure mixing of genes to create genetic diversity rather than exact copies of the parents. In meiosis, each germ cell splits twice so that each egg or sperm ends up with only one copy of each chromosome. Then when egg meets sperm, the embryo has the right number of chromosome pairs.

Before the first split occurs, the chromosomes in the germ cell pair up tightly and then each chromosome within a pair breaks and rejoins with pieces from its partner to exchange genes in a process called crossover. Then all these DNA breaks need to be rejoined quickly.

Think of a sandwich, study author and University of Michigan structural biologist Jayakrishnan Nandakumar explained. The ​“bun” is composed of four identical copies of a protein called MEILB2 on the top and bottom, with the two BRCA2 proteins between. The MEILB2 protein sandwich carries the BRCA2 protein precisely to the DNA break points.

It has long been surmised that BRCA2 has a link to infertility, and more information about the way germ cells are repaired (or not repaired) by these proteins as they divide could lead to insights about how infertility occurs.

“We know how the literature is rich with examples of BRCA2 mutations in cancer, but our findings now suggest that the MEILB2-binding region of BRCA2 might be a hotspot for discovering mutations related to infertility,” said Nandakumar.

To determine the structure of this BRCA2 complex, the researchers used X-ray crystallography at the Life Sciences Collaborative Access Team (LS-CAT) beamline at the APS. In this process, the protein crystal is bombarded with ultrabright X-rays. The patterns that are generated when the X-rays deflect off the atoms in the crystal allow the researchers to figure out where each atom is located in the 3D structure of the molecule, and in turn, figure out how the BRCA2 protein is connected to the MEILB2 protein.

Joseph Brunzelle, an assistant research professor at Northwestern University and staff member at LS-CAT, worked with the research team to obtain their X-ray data. As a testament to how far X-ray crystallography technology has come, Brunzelle said these scans were done in less than a minute, when the same work once took days to accomplish. (The speed of this type of research will be further improved by a massive upgrade to the APS, currently in progress.)

“Our techniques are well established, and we know how to make the beamline work efficiently,” Brunzelle said. ​“The APS delivers brilliant X-rays, and LS-CAT has state-of-the-art detectors and equipment, and a highly skilled staff. This research team also was able to get high-quality crystals of the BRCA2 complex, which yielded classical textbook diffraction that aided in the structure solution.”

Growing those crystals took much trial and error. Devon Pendlebury, a chemical biology graduate student at the University of Michigan, successfully crystallized the human form of the BRCA2 complex, and worked with LS-CAT in March of 2020.

From the X-ray crystallography data and additional experiments by University of Michigan graduate student Ritvija Agrawal, the team determined the structure of the protein complex and how the two proteins worked together. It was a somewhat unusual protein interaction, they reported.

To validate their findings, they created mutant versions of BRCA2 and MEILB2 based on their structure and showed how these mutants failed to form this complex with each other.

In further validation of the MEILB2-BRCA2 complex structure, collaborators at the University of Gothenburg in Sweden introduced equivalent mutant versions in mouse cells undergoing meiosis. Mutant BRCA2 or MEILB2 failed to get to the DNA breaks that needed to be rejoined.

“While we have known BRCA2 was necessary for DNA recombination in meiosis, we didn’t know how it was able to do this critical job efficiently,” Nandakumar said. ​“The MEILB2 that is part of this repair complex is only supposed to be present in cells that undergo meiosis but MEILB2 has also been found in several cancers. It may be that MEILB2 is very efficiently ​‘hijacking’ the BRCA2 in cancer cells, preventing proper repair of the DNA.”

Without other factors usually found in meiotic cells, the BRCA2 in these MEILB2-positive cancers might not get to the DNA breakpoints. Having a structure of this complex in hand, researchers may now find new approaches to regain BRCA2 function in MEILB2-positive cancers, Nandakumar suggests. And more study of this process could lead to better information about how it might be linked to infertility.