Making the right choice
LA JOLLA, Calif.—Scientists at the Salk Institute have found a microprotein that appears to help cells make big decisions that may avoid cancer. As they posit in a recent news release, cells face an important choice when facing a break in DNA: Is it better to do a task quickly and make mistakes, or to do it slowly but perfectly? Because, as they note, the decision matters, since the wrong choice could cause even more DNA damage and lead to cancer.
In work published in September in the journal Nature, Salk Institute scientists say they have unlocked a longstanding mystery about DNA repair, having found that a tiny protein called CYREN that helps cells choose the right pathway at the right time. This could offer future researchers a powerful tool to better guide them toward more effective treatments for cancer.
“Elucidating DNA repair pathways is critical to understanding how they can sometimes be toxic,” says Jan Karlseder, a professor in Salk’s Molecular and Cell Biology Laboratory and senior author of the new paper. “Our discovery of CYREN’s function not only adds to our body of knowledge, it gives us a new tool with which to potentially fight cancer.”
CYREN is a microprotein that inhibits a faster pathway to repair DNA when it can determine that a suitable DNA copy is available to be used for a slower pathway that is less prone to error and further damage. CYREN was initially discovered in 2015 by Salk scientist Alan Saghatelian as part of an effort to identify small proteins called “short ORF-encoded peptides” or SEPs, which are increasingly being found to have critical biological roles. But it is this recent discovery that shows CYREN’s potentially significant promise.
“We found a lot of these peptides in our earlier study, but we didn’t really know if any of them were important until the Karlseder lab got involved,” says Saghatelian, a professor in the Clayton Foundation Laboratories for Peptide Biology and one of the paper’s coauthors. “Thanks to this impressive new work, we now know there are some really important molecules among the hundreds we’re discovering.”
The most serious injuries that happen to DNA are known as double-strand breaks, wherein both strands are severed. This type of damage is particularly harmful because the repair process can lead to genome rearrangements. Such damage can be repaired by one of two pathways: a fast but error-prone process known as NHEJ (non-homologous end joining) or a slower, error-free pathway known as HR (homologous recombination).
The faster pathway efficiently rejoins broken strands, but in the case of multiple breaks it can join the wrong two ends together, making things much worse for a cell. The slower pathway is error-free because it relies on having an undamaged DNA sequence to guide the repair, but this means it can only operate after a cell has copied its genetic information in order to divide. Given that, the fast pathway operates exclusively before DNA is copied. What had baffled scientists was the fact that though the faster machinery is so efficient and prolific, it didn’t seem to outcompete the slower, more exact pathway after copying. Scientists have long suspected that something must be holding the faster option back in those cases.
Saghatelian’s research had suggested that CYREN was interacting with the master switch of the faster pathway, a protein called Ku. To determine the exact nature of the interaction, Karlseder’s team worked with a region of the genome where repair is normally suppressed to prevent dangerous fusions: the ends of chromosomes, called telomeres. Researchers can artificially disturb telomeres to activate the fast pathway, making it a model system to test CYREN’s effects.
“Telomeres offer a great research tool because they really need to repress repair, but there are ways to activate the repair machinery so that you can study it in a very controlled way,” explains Nausica Arnoult, a Salk research associate and first author of the paper. The Salk team did so, and found that with CYREN present, no repairs occurred after the cell copies its DNA, suggesting that it does flip off the master switch, Ku. Without CYREN around, Ku’s fast pathway was active both before DNA was copied and after.
Because the telomere experiments did not tell the team much about the competition between the fast and slow pathways, Arnoult next used molecular tools to compare repair in living cells with and without CYREN. She combined CRISPR with genes for fluorescent proteins that would be triggered by repair so that she could cut DNA in specific ways and see from the ensuing color which pathway had made the repair. She also analyzed all the protein interactions that took place.
These experiments revealed that CYREN directly attaches to Ku to inhibit the fast pathway both depending on timing (before or after DNA copying) and the type of DNA break (smooth vs. jagged, for example). Its activity can even tune the ratio of fast to slow repairs.
“Our study shows that CYREN is an important regulator of DNA-repair-pathway choice,” comments Karlseder, who holds the Donald and Darlene Shiley Chair at Salk. “The work also points to the exciting possibility of potentially introducing DNA damage in cancer cells and using CYREN to prevent them from making repairs.”
Salk is currently searching for other repair pathway choice regulators, based on the understanding that deregulating DNA repair in cancer could well lead to effective therapies. Because DNA repair is likely deregulated during aging, understanding these pathways might lead to a better understanding of the aging process and potentially to treatments for age-associated diseases as well. While a timeline for commercial applications is not yet possible, Karlseder confirms that further CYREN inhibitors have tremendous potential. “We are investigating whether there are correlations between CYREN expression and disease, and developing CYREN inhibitors. If successful, these can be tested in cellular laboratory research for their efficacy in synergizing with genotoxic drugs.”