No abstract

one of the authors regrets that she inadvertently omitted references to the computer program and protein potential function that the authors used for their simulations of barnase cited above. The following sentence should have been the first sentence of the Methods section: Molecular dynamics simulations were performed with the program ENCAD (44) and the potential energy function of Levitt et al. (45).

We have measured the rates and efficiencies of DNA unwinding (the number of ATP molecules hydrolyzed per DNA base pair unwound) catalyzed by the RecBC,RecBCD-K177Q (a site-directed mutant in the putative ATP-binding site in the RecD subunit), and RecBCD enzymes from Escherichia coli. The DNA unwinding rate was measured with a coupled assay in which unwound DNA is degraded by the combined action of the RecJ enzyme and exonuclease I. The rates of DNA unwinding by the RecBC and RecBCD-K177Q enzymes are reduced by about 4-fold compared to the case of the RecBCD enzyme. The efficiency of ATP hydrolysis was determined in two ways. First, it was calculated from the ratio of the ATP hydrolysis rate to the rate of DNA unwinding. In the second method, ATP hydrolysis was measured under conditions where all of the DNA substrate becomes completely unwound. The efficiency is the ratio of the total amount of ATP hydrolyzed to the amount of DNA substrate present in the reaction. The average efficiencies measured kinetically and by the complete unwinding experiment are as follows: 2.30 and 1.74 ATP/base pair (RecBCD enzyme); 1.44 and 1.28 (RecBC); and 1.20 and 1.07 (RecBCD-K177Q). The RecBC and RecBCD-K177Q enzymes are therefore able to couple ATP hydrolysis to DNA unwinding at least as efficiently as the RecBCD holoenzyme. The lower ATP per base pair ratios found for RecBC and RecBCD-K177Q indicate that the RecD subunit hydrolyzes ATP during DNA unwinding by the RecBCD enzyme.

The RecB and RecC subunits of the RecBCD enzyme from Escherichia coli were purified from cells containing plasmids overproducing these proteins [Boehmer, P.E., & Emmerson, P.T. (1991) Gene 102, 1-6]. RecB hydrolyzes ATP in the presence of either single- or double-stranded DNA. RecC stimulates ATP hydrolysis by RecB, particularly with double-stranded DNA. The steady-state kinetic parameters for ATP hydrolysis by RecBC with double-stranded DNA are kcat = 1600 min-1, Km = 8.1 microM, and kcat/Km(ATP) = 1.97 x 10(8) M-1 min-1. The RecBC enzyme acts processively, as measured by the effect of heparin on ATP hydrolysis stimulated by double-stranded DNA. About 2400 ATP molecules are hydrolyzed per enzyme bound to the end of a DNA molecule, using DNA substrates of 6250 or 21,400 base pairs. The enzyme is capable of unwinding a 6250 base pair double-stranded DNA molecule, in the presence of the single-stranded DNA binding protein of Escherichia coli. The steady-state kinetic parameters and the processivity are close to those found previously for the RecBCD-K177Q enzyme, with a lysine-to-glutamine mutation in the consensus ATP binding sequence in the RecD subunit, and are reduced compared to the RecBCD holoenzyme [Korangy, F., & Julin, D. A. (1992) J. Biol. Chem. 267, 1733-1740]. The most salient difference between RecBC and RecBCD-K177Q is the nuclease activity. RecBCD-K177Q produces a significant amount of acid-soluble DNA fragments from double-stranded DNA, while RecBC does not, even though the DNA does become unwound.

The RecBCD enzyme of Escherichia coli is an ATP-dependent DNA exonuclease and a helicase. Its exonuclease activity is subject to regulation by an octameric nucleotide sequence called chi. In this study, site-directed mutations were made in the carboxyl-terminal nuclease domain of the RecB subunit, and their effects on RecBCD's enzymatic activities were investigated. Mutation of two amino acid residues, Asp(1067) and Lys(1082), abolished nuclease activity on both single- and double-stranded DNA. Together with Asp(1080), these residues compose a motif that is similar to one shown to form the active site of several restriction endonucleases. The nuclease reactions catalyzed by the RecBCD enzyme should therefore follow the same mechanism as these restriction endonucleases. Furthermore, the mutant enzymes were unable to produce chi-specific fragments that are thought to result from the 3'-5' and 5'-3' single-stranded exonuclease activities of the enzyme during its reaction with chi-containing double-stranded DNA. The results show that the nuclease active site in the RecB C-terminal 30-kDa domain is the universal nuclease active site of RecBCD that is responsible for DNA degradation in both directions during the reaction with double-stranded DNA. A novel explanation for the observed nuclease polarity switch and RecBCD-DNA interaction is offered.