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Highly Processive DNA Repair Machine RecBCD May Be Regulated by AAA+ Superfamily
Location: 30
Mentor: Dr. Richard Myers
dsDNA breaks constitute some of the most severe damage an organism’s genome can endure. RecBCD, a heterotrimeric protein complex in Escherichia coli, functions as a highly processive helicase/nuclease crucial to repair these aforementioned dsDNA breaks through homologous genetic recombination. After the RecBCD complex binds to the end of a DNA break, nucleotide sequences in the E. coli genome called crossover hotspot instigators or Chi sequences (5'-GCTGGTGG-3') alter RecBCD nuclease activity, allowing for the recruitment of RecA protein, which drives homologous recombination. It is hypothesized that after completion of homologous recombination, the dissociation of the RecBCD subunits may be facilitated by three AAA+ family proteases: clpP, clpX, and lon. To test this hypothesis, this research focuses on creating gene fusions with the individual RecBCD subunits to tag them with HiBit, which, when bound to LgBit, reconstitutes the Luciferase enzyme, allowing detection and quantification through the bioluminescent signal it generates. The half-life of RecBCD will then be analyzed in the presence and absence of dsDNA breaks, RecA, clpP, clpX, and lon. The hypothesis predicts that the half-life of RecBCD’s individual subunits will increase in clpP-, clpX-, lon-, and RecA- conditions and in both the presence and absence of dsDNA breaks.
