Abstract
Coordinating multiple activities of complex enzymes is critical for life, including transcribing, replicating and repairing DNA. Bacterial RecBCD helicase–nuclease must coordinate DNA unwinding and cutting to repair broken DNA. Starting at a DNA end, RecBCD unwinds DNA with its fast RecD helicase on the 5′-ended strand and its slower RecB helicase on the 3′-ended strand. At Chi hotspots (5′ GCTGGTGG 3′), RecB’s nuclease cuts the 3′-ended strand and loads RecA strand-exchange protein onto it. We report that a small molecule NSAC1003, a sulfanyltriazolobenzimidazole, mimics Chi sites by sensitizing RecBCD to cut DNA at a Chi-independent position a certain percent of the DNA substrate's length. This percent decreases with increasing NSAC1003 concentration. Our data indicate that NSAC1003 slows RecB relative to RecD and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. ATP and NSAC1003 are competitive; computation docks NSAC1003 into RecB’s ATP-binding site, suggesting NSAC1003 acts directly on RecB. NSAC1003 will help elucidate molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes with activities coordinated at chromosomal sites.
Highlights
Complex, multi-subunit enzymes are required for most activities on nucleic acids, including replication, recombination, DNA repair, transcription, RNA splicing, and protein synthesis
Molecular docking [23] indicates that NSAC1003 binds to the ATPbinding site in RecB (Figure 5) with high affinity (0.3 μM) and with two consequences: the RecB helicase is slowed, likely reflecting the competition with ATP binding and hydrolysis, and the nuclease is sensitized to cut the DNA when RecD helicase stops, perhaps because NSAC1003 alters the RecBCD conformation in the same way as the two mutations in RecB (Y803H and V804E) [14]
These effects of NSAC1003 indicate that this compound will be informative in further elucidating Chi's control of RecBCD enzyme and other complex, multi-activity enzymes, as discussed below
Summary
Multi-subunit enzymes are required for most activities on nucleic acids, including replication, recombination, DNA repair, transcription, RNA splicing, and protein synthesis. Important is the repair of DNA double-strand breaks (DSBs) because when their DNA is broken, cells must repair it or they die. Faithful DSB repair requires DNA helicases and nucleases to prepare the broken DNA for interaction with an intact homologous DNA molecule. If the interacting DNA molecules differ genetically, DSB repair can produce genetic recombinants and thereby propel evolution. Understanding the molecular basis of DSB repair and recombination requires understanding how complex, multi-functional enzymes act on DNA. We describe a small organic molecule that alters the activity of a DNA helicase-nuclease complex in a novel way that lends new insights into the enzyme's mechanism and regulation
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