The 30-kDa C-terminal domain of the RecB protein is critical for the nuclease activity, but not the helicase activity, of the RecBCD enzyme from Escherichia coli.
The RecBCD enzyme from Escherichia coli is an ATP-dependent helicase and an ATP-stimulated nuclease. The 3' --> 5' exonuclease activity on double-stranded DNA is suppressed when the enzyme encounters a recombinational hot spot, called chi (chi). We have prepared a RecB deletion mutant (RecB1-929) by using results of limited proteolysis experiments that indicated that the RecB subunit consists of two main domains. The RecB1-929 protein, comprising the 100-kDa N-terminal domain of RecB, is an ATP-dependent helicase and a single-stranded DNA-dependent ATPase. Reconstitution of RecB1-929 with RecC and RecD leads to processive unwinding of a linearized plasmid. However, the reconstituted RecB1-929CD enzyme has lost the single-strand endo- and exonuclease and the double-strand exonuclease activities of the RecBCD enzyme. These results show that the 30-kDa C-terminal domain of RecB has an important role in the nuclease activity of RecBCD. On the basis of these findings, we propose the RecB C-terminal domain swing model to explain RecBCD's transformation from a 3' --> 5' exonuclease to a helicase when it meets a chi site.
Yu
M
M
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.
Souaya
J
J
Julin
D A
DA
D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D004265:DNA Helicases