This week we profile a recent publication in Nucleic Acids Research from
Dr. Gerald Smith (pictured) at the Fred Hutchinson Cancer Research Center
Can you provide a brief overview of your lab’s current research focus?
Our lab studies DNA break repair and genetic recombination in bacteria (E. coli) and fission yeast (S. pombe) using both genetic and biochemical methods. When their DNA is broken, cells must repair it or they die. Successful repair often employs homologous recombination, the generation of new combinations of gene alleles that provide the diversity for evolution to proceed efficiently. During sex cell formation (meiosis) DNA is programmed to be broken and then to be repaired to generate crossover connections between homologous chromosomes, which are important for proper chromosome segregation and viable progeny. Our lab thus studies both the formation and repair of DNA double-strand breaks (DSBs) in S. pombe. In E. coli our research is centered on the RecBCD helicase-nuclease – how it works and how inhibitors of it may provide a sorely needed, novel class of antibiotics.
Years ago, we showed how the three-subunit E. coli RecBCD helicase-nuclease unwinds DNA starting from a DNA double-strand break. Soon after that, we showed that during unwinding RecBCD cuts DNA at Chi recombinational hotspots (5’ GCTGGTGG 3’) to create a recombinogenic 3’ single-stranded DNA end. Further genetics and enzymology led to our “nuclease-swing” model, in which the RecB nuclease domain swings on its 19-amino-acid tether to move from an inactive “storage” site to the exit of a tunnel in RecC through which the Chi-containing strand is extruded during unwinding. Sue Amundsen tested this model by changing the length, the amino-acid composition, and the stiffness of the tether. Her results clearly show that the tether must be long enough and stiff enough, but not too stiff, for the nuclease to cut the DNA at Chi and to enhance recombination locally.
What is the significance of the findings in this publication?
Sue’s results support the nuclease-swing model, which is the most molecularly detailed model yet proposed for how a homologous recombination (and DNA break repair) hotspot works. Her mutant enzymes provide excellent material for direct physical tests of this model. Because nearly all bacteria have analogs of RecBCD important for DNA break repair, our results are likely to be widely applicable to studying DNA break repair in all bacteria. In fact, all enteric bacteria tested, such as Salmonella, Klebsiella, and Vibrio, which can cause serious human disease or death, have RecBCD enzymes that act at the same Chi sequence active in E. coli. Our lab’s search for novel antibiotics that inhibit RecBCD and its analogs will be enhanced by this additional knowledge of how DNA break repair, essential for all life, occurs.
What are the next steps for this research?
We plan to test our nuclease-swing model using physical assays, such as FRET (Förster resonance energy transfer). In our model, the nuclease domain swings on the tether about 5 nm, from the “storage” site to the tunnel exit. This is an ideal distance for FRET. But making the appropriate mutant enzymes carrying two fluorescent markers has been a problem. Meanwhile, Sue is making additional mutants, changing key amino acids at various points in the enzyme to further test our model genetically.
This research was funded by:
NIH research grant R35 GM118120; Fred Hutchinson Cancer Research Center “IN for the Hutch” funds; Bill and Melinda Gates Foundation Grand Challenges Explorations grant.
