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Copenhagen Team IDs Protein Complex Key to DNA Damage Repair

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NEW YORK (GenomeWeb) – A team led by researchers at the University of Copenhagen has identified a protein complex key to DNA damage repair (DDR).

Described in a study published this month in Cell, the complex, named shieldin, is involved in DDR processes including DNA end resection and non-homologous end joining. It also plays a role in sensitizing BRCA1-depleted tumor cells to PARP inhibitors, said Chunaram Choudhary, professor at the University of Copenhagen's Novo Nordisk Foundation Center for Protein Research and senior author on the paper.

Choudhary noted that he and his colleagues discovered the complex as part of a project researching interaction partners of the established DNA repair factors 53BP1, BRCA1, and MDC1. These proteins, he said, are known to play important roles in DNA repair, but their modes of action are not well characterized.

"When you look at the protein 53BP1, for instance, people have been studying this for the last 17 years," Choudhary said. "There are numerous papers that [look at] how this protein plays a role in DNA damage repair, and making different antibodies, and in sensitivities to drugs. But nobody really has a clue how it works."

One way to explore these proteins' mechanisms of action is to look at other proteins they interact with. MDC1 and 53BP1 have no enzymatic activity themselves, "so, obviously they must be interacting with something that is fundamentally very important," Choudhary said.

However, profiling the interactors of DDR proteins is complicated by the fact that these proteins are typically chromatin-bound, which means researchers must use harsh conditions to extract them for analysis. But these harsh extraction conditions can disrupt the native protein-protein interactions such experiments aim to explore in the first place.

To get around this problem, the Copenhagen researchers combined CRISPR gene editing with ascorbic acid peroxidase (APEX) labeling and quantitative mass spectrometry, which allowed them to identify interactors of the DDR proteins without having to co-purify them.

In APEX labeling, researchers genetically insert the APEX tag into their protein of interest. Upon stimulation with hydrogen peroxide, this tag releases biotin-phenoxyl radicals that tag nearby proteins in the cell, and these tagged proteins can then be pulled out of the sample using streptavidin-based enrichment and then analyzed using mass spec.

In the Cell study, the researchers inserted the APEX tag into the 53BP1, BRCA1, and MDC1 proteins and used mass spec to quantify the proteins pulled down in the tagged cells versus untagged controls, the idea being that proteins present at higher levels in the tagged samples compared to the controls were in close proximity to the DDR proteins of interest and therefore likely interactors.

The researchers determined roughly 90 percent of the proteins they identified to be background binders, leaving them with a set of roughly 400 proteins that were likely interactors. Of those 400 proteins, around 40 percent had previously been implicated in DDR, DNA replication, or telomere maintenance. Of the top 100 most highly enriched proteins for each of the three proteins investigated, more than 50 percent had been implicated in DDR by previous research.

"From a proteomics perspective, the networks we got were really amazingly clean and clear," Choudhary said. "For each of the proteins we got hundreds of interactions, but, nevertheless, the specificity [of these interactions] seems to be really amazing."

Among the top hits for 53BP1 was a previously uncharacterized protein the researchers named RINN1, which their networks predicted was also an interactor of USP28 and REV7, two proteins known to work with 53BP1 to repair double-strand DNA breaks.

After generating an antibody to RINN1 and confirming its expression in a variety of human cell lines, the researchers used affinity-purification mass spec to identify interactors of RINN1, finding that it interacted with the protein REV7 as well as with two other previously uncharacterized proteins, which they named RINN2 and RINN3.

Together, these proteins form what Choudhary and his colleagues termed the shieldin complex, a protein complex that they determined serves as an effector for 53BP1 that promotes DNA repair through the non-homologous end joining (NHEJ) process while suppressing repair via the more error-prone DNA end resection process.

This has potential clinical significance in that other components of the 53BP1 DDR pathway have been shown, when depleted, to lead to resistance to PARP inhibitions.

"We find that all three [RINN] proteins provide the exact same phenotype," Choudhary said. "So, from a cancer perspective it is very interesting to understand how this pathway actually provides resistance."

"You could conceive that if a patient has mutations in any of these genes, that would predict that [a PARP inhibitor] would not work for them," he added. "We think that in the future one could actually test the status of these genes for making the decision in the clinic whether a patient will benefit from [such a] drug or not. You could also use that same knowledge when drug resistance develops to understand whether any of these genes are responsible for that."

The work also sheds insight into a more basic biological question — the evolution of antibody class switching, which is the process by which B cells switch from production of one immunoglobulin type to another (for instance, from IgM to IgG). This involves DNA breakage and repair controlled by the 53BP1 pathway, but it has been unclear where in evolutionary history this process emerged. As potentially one of the last components in the process to appear, identification of the shieldin complex could help scientists more precisely date the emergence of class switching, Choudhary said.

Moving forward, he said, his lab hopes to further explore the biochemical and structural details of the shieldin complex. They also plan to use the CRISPR-APEX combination to study other proteins not amenable to conventional AP-MS approaches.

"We're very keen to use this to look for interactions of other proteins where we have similar problems in getting interactions otherwise," he said. "That's one thing that we are very excited about."