Biochemical characterization of P22 phage-modified Escherichia coli RecBCD enzyme.
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.
Murphy
K C
KC
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical Center, Worcester 01605.
D017100:Bacteriophage P22 / Q000201:enzymology*; D010582:Bacteriophage lambda / Q000378:metabolism; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004265:DNA Helicases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D017886:Durapatite; D004926:Escherichia coli / Q000201:enzymology*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017354:Point Mutation; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism / Q000494:pharmacology
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D014764:Viral Proteins; C065410:gam protein, Coliphage; D017886:Durapatite; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D004265:DNA Helicases