The nuclease domain of the Escherichia coli RecBCD enzyme catalyzes degradation of linear and circular single-stranded and double-stranded DNA.
The 30 kDa C-terminal domain of the RecB protein (RecB30) has nuclease activity and is believed to be responsible for the nucleolytic activities of the RecBCD enzyme. However, the RecB30 protein, studied as a histidine-tagged fusion protein, appeared to have very low nucleolytic activity on single-stranded (ss) DNA [Zhang, X. J., and Julin, D. A. (1999) Nucleic Acids Res. 27, 4200-4207], which raised the question of whether RecB30 was indeed the sole nuclease domain of RecBCD. Here, we have purified the RecB30 protein without a fusion tag. We report that RecB30 efficiently degrades both linear and circular single- and double-stranded (ds) DNA. The endonucleolytic cleavage of circular dsDNA is consistent with the fact that RecB30 has amino acid sequence similarity to some restriction endonucleases. However, endonuclease activity on dsDNA had never been seen before for RecBCD or any fragments of RecBCD. Kinetic analysis indicates that RecB30 is at least as active as RecBCD on the ssDNA substrates. These results provide direct evidence that RecB30 is the universal nuclease domain of RecBCD. The fact that the RecB30 nuclease domain alone has high intrinsic nuclease activity and can cleave dsDNA endonucleolytically suggests that the nuclease activity of RecB30 is modulated when it is part of the RecBCD holoenzyme. A new model has been proposed to explain the regulation of the RecB30 nuclease in RecBCD.
Sun
Jian-Zhong
JZ
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA.
Julin
Douglas A
DA
Hu
Jin-Shan
JS
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D002413:Cations, Divalent; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D043211:Exodeoxyribonuclease V / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009690:Nucleic Acid Conformation; D013997:Time Factors
D002413:Cations, Divalent; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D008670:Metals; D004247:DNA; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli