Until now, the DNA repair process — which proteins are involved and how they make it run — has been something of a black box: we know what goes in, but what happens on the inside is one big question mark.
In May, several geneticists at HHMI and Harvard opened up that box by presenting research in Science revealing a vast network of many hundreds of human and mouse proteins that are phosphorylated and called into action in response to DNA damage.
ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related), the two key protein kinases at the heart of the DNA damage response, were previously thought to have only 20 to 25 substrates.
“When we started, we didn’t expect to find too many more,” says Steve Elledge, one of the paper’s leading authors and a Howard Hughes investigator at Harvard’s Brigham and Women’s Hospital. “But what we found was really quite surprising, that we found hundreds more.” To be precise, 700 more, with a total of 900 regulated phosphorylation sites identified.
DNA damage is taken seriously by the cell. When genotoxic stress causes damage — typically in the form of chromosomal breaks or stalled replication — ATM and ATR are called upon to phosphorylate key proteins that in turn initiate various intracellular signaling cascades. “We knew that there was some basic machinery that worked to actually do the heavy lifting in terms of repairing DNA,” Elledge says. “What we didn’t know is that these are all probably being controlled by ATM and ATR.”
Using a combination of isotope labeling and immunoprecipitation, the team was able to isolate the phospho-SQ (serine-glutamine) and phospho-TQ (threonine-glutamine) sites of the ATM and ATR substrates. By taking advantage of the fact that it is difficult to create phospho-specific antibodies, they were able to capture more phospho-proteins. “Everybody makes antibodies for sites and they think they’re specific, but in fact, they’re not specific, especially for phospho-sites,” Elledge says. “We discovered that they weren’t specific, but then instead of saying they’re terrible, we said, ‘Oh, we can use this property to find more and more things that aren’t exactly the same, but are related.’”
In general, their findings open the door to a new world when it comes to understanding the cellular response to DNA damage. “The whole response to broken chromosomes and stalled replication is much deeper than we previously appreciated, and it involves almost every aspect of cellular physiology,” Elledge says. “So the cell really changes its physiology completely in response to these stresses.” The team found connections to other pathways that they didn’t expect: RNA processing, translational control, and the insulin signaling pathway.
As drug discovery goes, they may have created a very useful tool. For instance, screening for targets of ATM or ATR phosphorylation may lead to discovering candidate disease genes. “We can’t really say what the most important pathways are,” Elledge says. “What we’ve basically developed is this resource that everyone can use now to find out about their proteins.”