A swing and a swing and a hit: DNA control of RecBCD conformation and activity

Across organs and organisms, the loss of precise control of necessary biological functions can lead to detrimental consequences. Cancers stem from a loss of control of cell division, and autoimmune diseases from a loss of control of immune activation. 

This loss of control often occurs at the level of enzyme activity. Enzymes are molecular machines that allow living organisms to do everything from gleaning energy from food and replicating DNA to breaking down problematic proteins and signaling for cell death. Enzymatic function is, as a rule, tightly regulated so that enzymes function only when and where they are needed.

Understanding how enzymatic function is regulated is a central question across biological and biomedical research. For some enzymes, this is simply an on/off switch, but other enzymes are more complex and employ a wider breadth of control mechanisms.

One of the most complex enzymes known is the bacterial RecBCD, which has three protein subunits (RecB, RecC, and RecD) and a total of nine enzymatic activities, including DNA helicase (DNA unwinding) and nuclease (DNA cutting) functions. RecBCD facilitates homologous recombination, a process necessary for faithful repair after DNA damage and for genetic diversification. DNA unwinding and cutting allows the damaged DNA to match up with identical intact DNA as a template for rebuilding.

RecBCD’s activity is regulated by the Chi recombination hotspot, an eight nucleotide DNA sequence that controls the enzyme’s helicase and nuclease functions. In its first encounter with broken DNA, the enzyme does not cut (and thus potentially damage) DNA randomly but instead cuts at the Chi sequence when DNA repair or recombination is necessary and feasible. If the DNA cannot be repaired (for example if there is no matching DNA to use as a template or if other enzymes essential for recombination are missing), then in subsequent encounters RecBCD can cut the DNA into small pieces for recycling. “The mechanism of Chi’s regulation of RecBCD has yet to be completely defined,” said Dr. Gerry Smith in the Fred Hutch Basic Sciences Division. 

The Smith lab investigates how Chi so remarkably controls RecBCD enzymatic activity. Previous work has identified the primary functions of RecBCD’s three subunits: RecB is a helicase and a nuclease (in two separate domains connected by a tether, like two balls attached by a string), RecD is a helicase, and RecC binds the other two subunits and recognizes the Chi sequence. Their proposed model for Chi’s control of RecBCD function suggests that the initial binding interaction between RecBCD and broken DNA triggers a conformational change where the part of the RecB subunit that contains its nuclease activity swings away from the DNA, physically separating them to prevent random DNA cutting. RecB and RecD, each moving on a different DNA strand, unwind the DNA while RecC scans for the Chi sequence. Once RecC encounters Chi, RecD stops unwinding and the RecB nuclease swings back to the DNA and cuts it, allowing for homologous repair to proceed. Smith indicated, “We have identified 15 sites spread throughout the enzyme that are required for a complete response to the Chi sequence, supporting our inter-subunit signal transduction model for regulation of RecBCD.” The mechanism of exactly how these sites interact with Chi and with each other to confer strict control of RecBCD enzymatic function is the subject of their current research.

Researchers in the Smith lab noticed that the RecB tether that connects the RecB helicase and nuclease domains sits in a groove in the RecC subunit. In a recent study published in Genetics, they investigated this interaction in depth. 

The team created numerous enzyme variants that were mutated either along the RecB tether or RecC groove. They found that many mutations to these sites disrupted Chi’s control of the nuclease activity. Instead of cutting DNA and recombining at sites near Chi sequences, mutant enzymes cut and recombined DNA at sites spread out across the DNA genome, indicating that the RecB tether-RecC groove interaction is necessary for enzyme control by Chi.

Using protein-cutting enzymes called proteases to probe the exposure of the RecC groove on the RecBCD surface, the Smith lab found that the RecC groove is more susceptible to protease damage, and therefore more exposed, when RecBCD binds to DNA. This supports a model where the RecB tether sits in the RecC groove in the absence of DNA but swings out of place when DNA is bound. “Swinging would sequester the nuclease domain away from DNA preventing rampant degradation that could limit cellular survival until Chi is encountered,” Smith commented. Put simply, a binding interaction leads to structural changes that restrain enzyme function.

RecBCD provides an example of how binding interactions, subunit communication, and conformational changes can all be used as methods to direct and limit when and where enzyme function occurs. Understanding mechanisms of enzyme control provides a basis for uncovering how other complex enzymes, such as human DNA and RNA polymerases, are regulated, and how loss of proper regulation can lead to disease. 

Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Member Dr. Gerry Smith contributed to this research.

The spotlighted research was funded by the National Institutes of Health.

Amundsen SK, Zhu Y, Smith GR. 2026. Chi hotspot control of RecBCD enzyme requires a RecB tether - RecC groove crosspoint interaction. Genetics. doi: 10.1093/genetics/iyaf240