Taking a swing at a fixed arm’s length

From the Smith lab, Basic Sciences Division

A Chi site or Chi sequence is a short stretch of DNA in the bacterial genome near which homologous recombination is more likely to occur than on average across the genome. The sequence of the Chi site is unique to each group of closely related organisms. In Escherichia coli (E. coli) and other enteric bacteria, such as Salmonella, the core sequence is 5’-GCTGGTGG-3’, but Chi activity is strongly influenced by 4 to 7 nucleotides on the 3' side of the core sequence. Originally discovered in the genome of bacteriophage lambda, a virus that infects E. coli, Chi sites occur about 1000 times in the E. coli genome.

Chi sites stimulate DNA double-strand break repair in bacteria; breaks can arise from radiation or chemical treatments, or from replication fork breakage during DNA replication. Chi does so by altering the conformation of the RecBCD helicase-nuclease, triggering a major change in the activities of this enzyme. Upon encountering the Chi sequence as it unwinds DNA from a double-strand end, RecBCD cuts the DNA a few nucleotides to the 3’ side of Chi, within the important sequences noted above. The resulting 3’ single-stranded DNA (ssDNA) is bound by multiple molecules of RecA protein that facilitate "strand invasion," in which one strand of a homologous double-stranded DNA is displaced by the RecA-associated ssDNA. During repair of the broken DNA, recombinant DNA molecules can be generated if the two interacting DNAs are genetically different.

How can a short DNA sequence control a huge (330 kDa) helicase-nuclease enzyme?  The lab's previously published structures of RecBCD bound and unbound to DNA and the crystal structure from another lab led to the nuclease-swing model.  In this model the nuclease activity is regulated by the 32 kDa nuclease domain swinging, after the enzyme meets Chi, on a 19-amino-acid tether that connects its nuclease and helicase domains.  Intrigued by this possibility, Basic Sciences Member Dr. Gerry Smith, and a staff scientist in his laboratory, Dr. Susan Amundsen, set out to test if the length and flexibility of the tether may be important in the control of RecBCD by Chi. Their work was published in a recent issue of Nucleic Acids Research.

Nuclease-swing model for Chi’s control of RecBCD enzyme Figure from publication

To test the importance of the tether, the authors carried out an extensive mutational analysis of the tether and tested the activities of RecBCD in cells. Recombination proficiency and hotspot activity were assayed using a standard hotspot cross of two lambda phages.  They found that shortening the tether by even one amino acid (or up to 19 amino acids) significantly (or almost completely) reduced Chi-dependent RecBCD activities. The shortened tethers interfered with Chi activity but not with general nuclease activity.  Since the tether appears fully stretched out in published structures, the authors wondered if a longer tether might be functional and result in greater Chi hotspot activity.  Interestingly, lengthening the tether by 19 or 38, but not by 9 or 10, amino acids reduced Chi-dependent RecBCD function.  The authors further showed that while the exact amino acid sequence of the tether is not crucial, substitutions to impact the flexibility of the tether dramatically reduce Chi-dependent RecBCD activities.  The wild-type tether has only one proline and one glycine residue; proline residues have more limited rotational capacity, while glycine residues have a greater rotational capacity.  Mutants containing more proline or glycine residues than wild type showed that changing the flexibility of the tether strongly affects the ability of RecBCD to respond to Chi and promote recombination. The enzymatic activities of RecBCD extracted from the mutant cells were consistent with the genetic activities of the tether mutants.

Using both a genetic and biochemical approach, Drs. Amundsen and Smith show evidence that supports the nuclease-swing model for the control of RecBCD enzyme by Chi recombination hotspots.  In this model, upon encountering Chi, the nuclease domain of RecBCD is triggered to swing on its 19-amino-acid tether into a position where it can cut DNA near Chi and begin loading RecA strand-exchange protein onto the newly created end.  “RecBCD is an example of a powerful nuclease, whose activity has to be regulated so that DNA can be repaired at the proper time.  It can’t be a rampant nuclease; otherwise, that will create havoc in the cell” said Dr. Amundsen.  “Having the crystal structure of the enzyme has helped tremendously.  We know where all the parts of RecBCD, including the nuclease site and the tether, are. The structure has been huge” commented Dr. Amundsen, who explained what enabled her to genetically test the nuclease-swing model.  As she showed, changing the tether can strongly impact, even abolish, Chi activity, demonstrating the essential role of the tether. Dr. Smith, who started studying Chi 42 years ago, remarked on the amazing features of this system: “DNA with multiple Chi sites is cut near Chi only once per DNA end, which tells us there is remarkable control.  We don’t know how this happens, but perhaps it's got to do with a further rotation of the nuclease domain after it swings.”  Indeed, there is much to uncover, and technological advances have opened more doors. “First we had genetics, then came the sophistication of biochemical assays.  We merged those two into molecular biology, and now we are heading into the future of atomic biology” enthused Dr. Smith.

Amundsen SK,Smith GR. 2018. The RecB helicase-nuclease tether mediates Chi hotspot control of RecBCD enzyme. Nucleic Acids Res. 47(1): 197-209

Funding was provided by the National Institutes of Health.