The mutant recBCD enzyme, recB2109CD enzyme, has helicase activity but does not promote efficient joint molecule formation in vitro.
The Escherichia coli recB2109CD enzyme displays a defect in homologous recombination. In vitro, it possesses significant levels of non-specific nuclease activity but is deficient in chi-dependent nicking activity. To determine whether an alteration in helicase activity contributes further to its in vivo defect, the ability of recB2109CD enzyme to unwind dsDNA was examined. The mutant enzyme is able to unwind DNA but has a kcat which is one-third that of the wild-type enzyme. While the Km for DNA ends of the wild-type and mutant enzymes at low NaCl concentrations are essentially equivalent, the Km for ATP of recB2109CD enzyme is nearly six times greater. The processivity of unwinding (i.e. the average length of DNA unwound before recB2109CD enzyme dissociates from the DNA substrate) at 1 mM-Mg2+ ion and 1 mM-ATP is approximately 13 kb/end, whereas that of wild-type recBCD enzyme is 30 kb/end. In an assay which requires the co-ordinate actions of the recBCD, recA, and SSB proteins, joint molecule formation in the presence of recB2109CD enzyme is up to sixfold slower and proceeds to a lower extent than that mediated by the wild-type enzyme. We conclude that although the reduced helicase activity of the mutant recBCD enzyme may contribute to its recombination deficiency, its defect in the chi-dependent attenuation of non-specific nuclease activity is primarily responsible for the recombination-deficiency of E. coli strains bearing the recB2109 mutation.
Eggleston
A K
AK
Department of Cell, Molecular & Structural Biology, Northwestern University Medical School, Chicago, IL 60611.
Kowalczykowski
S C
SC
D000255:Adenosine Triphosphate / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009154:Mutation; D012965:Sodium Chloride / Q000494:pharmacology
D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D004247:DNA; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D004265:DNA Helicases; D008274:Magnesium