Science Spotlight

Swing and a hit! RecB contacts RecC to repair DNA breaks

From the Smith Lab, Basic Sciences Division

Breaks in our genetic code termed “DNA double-strand breaks” create cellular stress and cell death if not rapidly repaired. Environmental mutagens including UV light can cause DNA breaks, and these breaks tend to occur more frequently in DNA replication and transcription intermediates. The bacterium Escherichia coli expresses the RecBCD protein with both DNA-unwinding (helicase) activity and DNA-cutting (nuclease, or Nuc) activity domains, that efficiently repair DNA breaks. The Smith Lab in the Basic Sciences division at Fred Hutchinson Cancer Center has extensively dissected the mechanism of Chi site-dependent DNA repair that is facilitated by RecBCD. The Chi site is a specific 8-nucleotide sequence that the RecC subunit recognizes and RecB Nuc cuts to allow for replacement of DNA-containing breaks with the intact form of DNA through a process called homologous recombination. The role of RecB Nuc to cut the DNA is regulated by the position of this subunit. The researchers have shown that upon DNA binding to RecBCD, the RecB Nuc domain swings from its docked site to a new site, likely on the opposite side of RecC subunit. A second swing of RecB Nuc returns the domain back to the exit tunnel where Nuc cuts the DNA as it exits the RecBCD enzyme. “We sometimes think of the RecB nuclease [Nuc] domain swinging on its 19-amino-acid tether like a dog on a leash – it must move from one side to another to behave properly,” stated Dr. Gerry Smith. “The nuclease must respond to Chi, by moving and cutting the DNA, like a dog must respond to the master’s whistle and yank on the leash.” As the Nuc domain swings into action, it is not clear what residues on the RecC surface contact the RecB Nuc domain and if this interaction is necessary for efficient Chi-dependent DNA repair. The Smith lab used many RecBCD mutants to identify RecB and RecC contact sites and to determine the role of these residues during DNA break repair. Their findings were published recently in Genetics.

RecBCD has three subunits: RecB, RecC, and RecD depicted below as a cartoon and crystal structure. From analyzing the wild-type RecBCD crystal and cryoEM structures, the researchers noticed a flexible loop on the surface of RecC that could provide a dynamic docking site for RecB. Deletion of the RecC loop significantly reduced Chi-dependent DNA repair activity but did not completely block it. This discovery highlighted this loop structure as a key determinant of Chi’s control of RecBCD-mediated repair of DNA breaks.

The E. coli RecBCD helicase-nuclease protein is depicted on the left in cartoon version with the subunits, and as a crystal structure (left images). The model of RecB Nuc domain swinging is depicted with red arrows (right images). Upon DNA binding, Nuc makes its first swing to contact the RecC loop (+DNA image). After a Chi site is encountered during DNA unwinding, the second swing of Nuc occurs to return the Nuc subunit to its original position and site of Nuc-mediated cutting of the DNA at Chi (after Chi image). The crystal structure for DNA bound RecBCD is known (after Chi image) and the other images are inferred crystal structures.
The E. coli RecBCD helicase-nuclease protein is depicted on the left in cartoon version with the subunits, and as a crystal structure (left images). The model of RecB Nuc domain swinging is depicted with red arrows (right images). Upon DNA binding, Nuc makes its first swing to contact the RecC loop (+DNA image). After a Chi site is encountered during DNA unwinding, the second swing of Nuc occurs to return the Nuc subunit to its original position and site of Nuc-mediated cutting of the DNA at Chi (after Chi image). The crystal structure for DNA bound RecBCD is known (after Chi image) and the other images are inferred crystal structures. Image modified from figure in published article

The researchers took two approaches to determine the points of contact between the RecB Nuc domain and RecC after Nuc swinging (+DNA image). First, they used a modeling program ClusPro to computationally predict the amino acids important for RecB docking onto RecC. Modeling-based predictions of docking sites were generated for wild-type RecBCD and several subunit combinations, including at minimum RecB Nuc and RecC subunits. These models highlighted the same four amino acids among the top candidates. The four sites were validated as functionally important for Chi-dependent recombination activity by genetically altering each codon to alanine or reversing the amino acid charge of contact sites of either RecB or RecC, or for both.

The second method used to identify RecB and RecC contact points upon DNA binding was to randomly induce mutations into the recC gene at sites within and flanking the RecC loop region (amino acids 193 to 346 in the 1,120 amino acid RecC subunit). This approach identified an additional site, tryptophan (W) at codon 248, with significant Chi-dependent recombination activity that can be restricted with codon change to arginine (R) or more potently limited for the lysine (K) substitution. Curiously, RecC amino acid W248 is not within the RecC loop structure but instead is situated at an internal site just behind the loop that also contacts another residue of RecC that is oriented adjacent to the loop. Additional structural studies of W248R and W248K mutants may provide interesting insights into the conformational changes of RecC at different points of the signal transduction pathway to repair DNA breaks. Together, “we test and find support for our nuclease-swing model for RecBCD DNA repair enzyme,” stated Dr. Smith. “We predicted, from previous results, that a region on the surface of RecBCD is important for RecBCD to respond to Chi during the enzyme’s unwinding of DNA. This region is a flexible loop of about 40 amino acids” and “is essential for full Chi activity but is dispensable for unwinding and general nuclease activity” as “accounted for by our model.”

Future questions that Dr. Smith intends to address include the following, “What are the conformational changes of RecBCD upon binding DNA, during unwinding, and upon acting at Chi? Are these changes altered as we expect by the mutants described in our paper?” Dr. Smith continued, “We expect that our genetic analysis reported in this paper will lead to direct physical assays, such as cryoEM and FRET, to further test our nuclease-swing model.”

Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member Gerald Smith contributed to this work.

The spotlighted research was funded by the National Institute of General Medical Sciences, USA and National Institutes of Health, USA.

Amundsen SK, Richardson A, Ha K, Smith GR. 2022. A flexible RecC surface loop required for Chi hotspot control of RecBCD enzyme. Genetics. iyac175. Online ahead of print.