Science Spotlight

Is that a Chi site? How to fool a smart enzyme

From the Smith Laboratory, Basic Sciences Division

The DNA of all organisms, be it bacteria or humans, is constantly bombarded with damage either by environmental mutagens such as the exposure to UV light or because of erroneous replication. The survival of the organisms depends on efficient repair of damaged or broken DNA. Homologous recombination is a mechanism of DNA repair of broken DNA via interaction with an intact homologous DNA molecule thereby ensuring faithful repair. In instances where the DNA molecules are genetically different, the repair process results in genetic recombinants and this heterogeneity propels evolution. When this process goes awry, it can lead to pathologies such as miscarriage, developmental disorders or cancer.

The Smith laboratory (Basic Science Division) has long studied the major mechanism by which bacteria repair breaks in their DNA that naturally occur during processes such as chromosome replication. In the bacterium Escherichia coli (E. coli), homologous recombination is mediated by the RecBCD helicase–nuclease enzyme complex. The enzyme complex is composed of three different subunits called RecB, RecC, and RecD, hence the name. 

Early work by Dr. Gerry Smith and colleagues elucidated how the bacterial RecBCD both unwinds DNA from a broken end and cuts it at special sites known as "hotspots" of recombination. These hotspots are known as Chi (crossover hotspot instigator) with a defined nucleotide sequence 5’-GCTGGTGG-3’ that occurs about 1000 times in the E. coli genome. The lab went on to characterize the molecular determinants of Chi recombination hotspots and identified RecC mutants that disrupt the recognition of the Chi sequence but do not compromise the overall structure or activity of the RecBCD complex.

In a new study published in Nucleic Acids Research, Dr. Smith and colleagues characterized a small molecule inhibitor of RecBCD. The goal was to identify novel antibiotics that can disrupt this essential enzyme in bacteria thereby rendering the pathogen unable to fix DNA breaks caused by host defenses. The lab collaborated with Dr. Ryan Cirz at Achaogen, Inc., to find such compounds. “During a search for novel antibiotics, our collaborator Ryan Cirz found a compound NSAC1003 that inhibits the bacterial DNA repair enzyme RecBCD.  Ahmet Karabulut in our lab found that this compound has the remarkable ability to mimic the bacterial recombination hotspot Chi, at which RecBCD cuts DNA at high frequency to promote DNA repair and genetic recombination,” said Dr. Smith.

The reported compound (NSAC1003) mimics two RecBCD mutations that make RecBCD cut DNA at novel positions much like the uninhibited, wild-type enzyme cuts at Chi hotspots of genetic recombination. “Some years ago, Sue Amundsen and others in the lab (Amundsen et al., Genes Dev 2007) found two mutations at the RecB site that hydrolyzes ATP, the fuel for the unwinding motors. The mutations make RecB cut the DNA when RecD gets to the DNA end.” Dr. Smith explained. “We are pretty sure that NSAC1003 binds to the sites at RecB, but it is still unknown how these mutations and NSAC1003 make RecBCD behave so remarkably,” he added. 

First the authors assayed RecBCD activity upon incubation with increasing concentrations of NSAC1003. They demonstrated that NSAC1003 inhibits RecBCD Chi-independent nuclease activity in an ATP-competitive manner. The authors then used a computational docking method to define NSAC1003 binding site. Reassuringly, NSAC1003 was predicted to bind tightly to the RecB ATP site, thereby confirming that NSAC1003 binding to RecB is ATP-competitive. Dr. Smith further explained: “Ahmet showed that when RecD, the faster of RecBCD’s two DNA unwinding subunits, gets to the end of linear DNA, it signals the slower RecB unwinding subunit to cut the DNA where it is at that moment but only if NSAC1003 is present.  The more compound present, the slower RecB is and the closer to the starting DNA end are the cuts.”

A schematic of the "signal transduction model" for how Chi hotspots of genetic recombination control the RecBCD helicase-nuclease to repair broken DNA.  As proposed, NSAC1003 sensitizes RecBCD to cut DNA by bypassing steps 1 and 2 above and making RecD signal RecB (step 3) when RecD gets to the end of its DNA strand and necessarily stops (as in step 2 when Chi is present).
A schematic of the "signal transduction model" for how Chi hotspots of genetic recombination control the RecBCD helicase-nuclease to repair broken DNA. As proposed, NSAC1003 sensitizes RecBCD to cut DNA by bypassing steps 1 and 2 above and making RecD signal RecB (step 3) when RecD gets to the end of its DNA strand and necessarily stops (as in step 2 when Chi is present). Image courtesy of Gerry Smith.

When RecBCD binds to a DNA break, it unwinds the DNA with its fast RecD helicase on the 5’-ended strand and its slower RecB helicase on the 3’-ended strand, a process driven by ATP hydrolysis. When RecBCD encounters a Chi hotpot, RecB cuts the strand with the Chi sequence and loads the strand exchange factor RecA. Prior to this study, the molecular mechanism whereby a Chi hotspot signals RecBCD to cut DNA at Chi was unclear. Dr. Smith and colleagues work supports a signal transduction model for the Chi-RecBCD interaction (summarized in the schematic above). The effect of NSAC1003 on RecBCD bolsters this model by showing an additional way in which the enzyme can be activated to cut DNA, an early step in repair of broken DNA and making genetic recombinants.

Going forward, the Smith lab seeks to further elucidate the molecular mechanisms of RecBCD-Chi regulation and DNA repair. “One immediate goal is to see if NSAC1003 changes the conformation of RecBCD by using cryoEM (with Dr. Melody Campbell, now at Fred Hutch),” said Dr. Smith. “Another goal is to test more RecBCD inhibitors, some provided by our new collaborator Tom Lanyon-Hogg at the University of Oxford,” he added. In the long term, Dr. Smith hopes that this line of research will guide the development of new antibiotics, more and more crucial as bacteria become resistant to currently used regimens.  

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

This study was made possible by support from the National Institutes of Health.

Karabulut AC, Cirz RT, Taylor AF, Smith GR. 2020. Small-molecule sensitization of RecBCD helicase–nuclease to a Chi hotspot-activated state, Nucleic Acids Research, doi: 10.1093/nar/gkaa534