title: Isolation and characterization of the C-terminal nuclease domain from the RecB protein of Escherichia coli.
abstract: The RecB subunit of the Escherichia coli RecBCD enzyme has been shown in previous work to have two domains: an N-terminal 100 kDa domain with ATP-dependent helicase activity, and a C-terminal 30 kDa domain. The 30 kDa domain had nuclease activity when linked to a heterologous DNA binding protein, but by itself it appeared unable to bind DNA and lacked detectable nuclease activity. We have expressed and isolated this 30 kDa domain, called RecB(N), and show that it does have nuclease activity detectable at high protein concentration in the presence of polyethylene glycol, added as a molecular crowding agent. The activity is undetectable in a mutant RecB(N)protein in which an aspartate residue has been changed to alanine. Structural analysis of the wild-type and mutant RecB(N)proteins by second derivative absorbance and circular dichroism spectroscopy indicates that both are folded proteins with very similar secondary and tertiary structures. The results show that the Asp-->Ala mutation has not caused a significant structural change in the isolated domain and they support the conclusion that the C-terminal domain of RecB has the sole nuclease active site of RecBCD.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.
authors.forename: X J
authors.initials: XJ
authors.lastname: Zhang
authors.forename: D A
authors.initials: DA
authors.lastname: Julin
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D014764:Viral Proteins
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; C027729:helix-destabilizing proteins; D011092:Polyethylene Glycols; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D004265:DNA Helicases