Biochemical characterization of P22 phage-modified Escherichia coli RecBCD enzyme
Department of Molecular Genetics and Microbiology
Bacteriophage P22; Bacteriophage lambda; Chromatography; Chromatography, DEAE-Cellulose; Chromatography, Gel; DNA Helicases; DNA-Binding Proteins; Durapatite; Escherichia coli; Exodeoxyribonuclease V; Exodeoxyribonucleases; Kinetics; Macromolecular Substances; Mutagenesis, Site-Directed; Plasmids; Point Mutation; Substrate Specificity; Viral Proteins
Life Sciences | Medicine and Health Sciences
The biochemical properties of phage P22 Abc-modified RecBCD enzyme from Escherichia coli have been examined. RecBCD purified from a cell that expresses Abc (anti-RecBCD) contains all three RecBCD subunits and the 11.6-kDa Abc protein in equal stoichiometric amounts. Abc depresses the rate of RecBCD double-stranded DNA exonuclease, helicase, and ATPase activities about 3-4-fold, yet it has no effect on the rate of the single-stranded DNA exonuclease activity. Abc induces a slight increase in the ATP-independent single-stranded DNA endonuclease activity and does not induce dimerization of the RecBCD trimer. Abc-modified RecBCD helicase activity possesses reduced but significant processivity (10 kilobase pairs) relative to the native enzyme (30 kilobase pairs). In the absence of ATP, Abc-modified RecBCD shows a 2-4-fold higher affinity for double-stranded DNA ends. The RecBCD-binding Gam protein from bacteriophage lambda inhibits binding of both native and Abc-modified RecBCD to double-stranded DNA ends. Finally, unlike the native enzyme, the nonspecific nuclease activity of Abc-modified RecBCD is not suppressed by Chi sites in vitro. These findings are discussed in terms of the recombination-deficient phenotype of cells expressing Abc in vivo and the relationship between Abc-modified RecBCD and two mutant RecBCD's previously characterized: the RecBCD-K117Q and RecB2109CD mutant enzymes.
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Citation: J Biol Chem. 1994 Sep 9;269(36):22507-16.
The Journal of biological chemistry
Murphy, Kenan C., "Biochemical characterization of P22 phage-modified Escherichia coli RecBCD enzyme" (1994). Open Access Articles. 819.