TY - JOUR
T1 - The mutant recBCD enzyme, recB2109CD enzyme, has helicase activity but does not promote efficient joint molecule formation in vitro
AU - Eggleston, Angela K.
AU - Kowalczykowski, Stephen C.
PY - 1993
Y1 - 1993
N2 - 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 χ-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 concentration 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 χ-dependent attenuation of non-specific nuclease activity is primarily responsible for the recombination-deficiency of E. coli strains bearing the recB2109 mutation.
AB - 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 χ-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 concentration 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 χ-dependent attenuation of non-specific nuclease activity is primarily responsible for the recombination-deficiency of E. coli strains bearing the recB2109 mutation.
KW - ATP-dependent nuclease
KW - DNA helicase
KW - Genetic recombination
KW - recBCD enzyme
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M3 - Article
C2 - 8390578
AN - SCOPUS:0027296790
VL - 231
SP - 621
EP - 633
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 3
ER -