Taking the long way ‘round: the circuitous molecular route to DNA repair

From the Smith Lab, Basic Sciences Division

DNA holds great power within the cell. The information it encodes contains crucial and precise instructions to ensure the smooth operation of the processes essential for life. Thus, all cells rely heavily on the accuracy of that information. Yet along with this responsibility comes a heavy burden: one critical error in the code, one errant instruction, can spell disaster. Throughout their lives, our cells maintain a tenuous balance: environmental toxins, and the processes of life itself, conspire to mutate and corrupt the genetic code, while DNA guardians within the cell work tirelessly to prevent or correct such defects. Among the most dangerous damage that can occur are DNA breaks. Like tiny scissors, radiation, UV light, and certain drugs, as well as unavoidable errors during cell division, can snip the DNA strand in two, causing mutations that may lead to cancer or cell death. When this happens, a group of proteins within the cell leap to action to put the pieces back together.  But even this process is fraught. “If not properly regulated, DNA repair can fail and produce incomplete or rearranged chromosomes leading to sickness or death,” said Dr. Gerry Smith, a professor in the Basic Sciences Division at Fred Hutch.  In a recent paper, published in the journal Scientific Reports, Dr. Smith’s lab takes a closer look at how the DNA repair machinery recognizes and repairs DNA breaks.

In bacteria, broken DNA is repaired by the tripartite RecBCD protein complex. After a double-strand DNA break occurs, the complex binds the broken end. Two proteins in the complex (RecB and RecD) motor along the DNA, unwinding it while the third protein (RecC) watches for a nearby special DNA sequence known as Chi. Once the Chi site has been found, RecB then cuts the DNA and begins the process of stitching the broken ends back together.  The mystery lies in how these three proteins communicate to carry out this orchestrated series of events. Specifically, the sites of Chi recognition and subsequent DNA cutting reside on different proteins (RecC and RecB, respectively), and quite far apart (about 25 angstroms). How, then, does RecC communicate to RecB that it has found Chi and that it is time to make the cut?

structure of the RecBCD protein complex
Structure of the RecBCD protein complex

This information is communicated via conformational changes – shifts in the shape of the protein complex that indicate Chi has been recognized. To understand this process better, Dr. Smith’s lab, led by staff scientist Dr. Sue Amundsen, began examining mutations in these proteins. By examining the structure of the complex and locating the positions of mutations that block the physical transmission of the message, they reasoned, they could identify the molecular path it travels through the complex. Much to their surprise, the first such mutation they found was 24-42 angstroms from the Chi-binding site and a full 65 angstroms from the DNA cutting site. In other words, farther from either of these sites than they are from each other, and thus seemingly indicating that the message is being sent in quite the wrong direction. The authors then studied additional mutants – 63 in total – and found 5 points in the complex need to transmit the signal. By tracing a path through these 5 points, the authors found the sum total distance traveled from the Chi recognition site in RecC to the DNA cutting site in RecB to be a whopping 185 angstroms. An unexpectedly circuitous route to send a message between two sites that sit a mere 25 angstroms apart. Dr. Smith analogized the journey aptly: “This is like telephoning from Seattle to Denver to San Francisco to Bellevue to tell someone there to turn down the TV.  But maybe there are people in Denver and San Francisco who need to know (or who have control over the person in Bellevue).”

Protein complexes are involved in many important processes in the cell. Dr. Smith hopes that this work serves as a model to understand how proteins in other complexes work together to carry out complex functions. “I think the most important point of this paper is the large set of mutants that Sue made and analyzed to get genetic evidence for this major conformational change of RecBCD at Chi…we hope that others will use the approach she used to analyze other complex enzymes that move along DNA and change their activity at special sites, such as RNA and DNA polymerases.” He also believes there is much more to be learned about the process of DNA repair by examining the mutants generated in this study. “Meanwhile, a postdoc in our lab, Yihua Zhu, is purifying some of Sue’s mutants for direct cryoEM analysis by our new colleague [Dr.] Melody Campbell,” Dr. Smith said.

Amundsen S.K., Taylor A.F., Smith G.R. Chi hotspot control of RecBCD helicase-nuclease by long-range intramolecular signaling. Science Reports 10, 19415 (2020). https://doi.org/10.1038/s41598-020-73078-0

This work was supported by the NIH (grants R35 GM118120 to G.R.S. and P30 CA015704).

Fred Hutch/UW Cancer Consortium member Gerald Smith contributed to this work.