DNA Repair Protocols

Volume 152 of the series Methods in Molecular Biology™ pp 91-105

Detection and Quantitation of RecBCD Enzyme (Exonuclease V) Activity

  • Douglas A. JulinAffiliated withDepartment of Chemistry and Biochemistry, University of Maryland

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The RecBCD enzyme serves two functions in the bacterial cell: it is a nuclease that destroys linear double-stranded DNA (dsDNA), and a DNA helicase that generates single-stranded DNA (ssDNA) used by RecA protein to initiate homologous recombination (13). A specific DNA sequence called Chi (5′-GCTGGTGG) is a signal that regulates these two functions (1,3). An encounter with Chi by RecBCD during its reaction with dsDNA leads to suppression of the nuclease activity and reveals the recombination-initiating function of RecBCD (1,3). The enzyme in Escherichia coli and other bacteria consists of three protein subunits encoded by the recB, recC, and recD genes, whereas some bacteria (e.g., Bacillus subtilis, Lactococcus lactis) produce a two-subunit enzyme [AddAB and RexAB, respectively (4,5)]. The genes encoding these latter enzymes complement an E. coli recBCD deletion mutation in vivo (5,6), and the purified AddAB enzyme has similar catalytic activity to the more extensively studied E. coli enzyme (7).