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 Ï. 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 Ï-specific fragments that are thought to result from the 3â²-5â² and
5â²-3â² single-stranded exonuclease activities of the enzyme during its reaction with Ï-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.
The recombinational hot spot chi modulates the nuclease and helicase activities of the RecBCD enzyme, leading to generation of an early DNA intermediate for homologous recombination. Here we identify ...the subunit location of the nuclease active site in RecBCD. The isolated RecB protein cleaves circular single-stranded M13 phage DNA, but RecB1-929, comprising only the 100 kDa N-terminal domain of RecB, does not. We reported previously that the reconstituted RecB1-929CD enzyme also is not a nuclease, suggesting that the C-terminal 30 kDa domain of RecB is a non-specific ssDNA endonuclease. However, we were unable to detect nuclease activity with the subtilisin-generated C-terminal 30 kDa fragment of RecB. Since the subtilisin-generated fragment did not bind to a ssDNA-agarose column, we designed a chimeric enzyme by attaching the C-terminal 30 kDa domain of RecB to the gene 32 protein of T4 phage, a ssDNA binding protein that does not have strand scission ability. In addition, Asp427 in the chimeric enzyme (Asp1080 in RecB), a residue that is conserved among several RecB homologs, was substituted to alanine (the D427A mutant). The wild-type chimeric enzyme cleaves the M13 DNA and the D427A mutation abolishes the endonuclease activity of the chimeric enzyme but does not affect its DNA binding ability. This finding indicates an unusual bipartite nature in the structural organization of RecB, in which the DNA-binding function is located in the N-terminal 100 kDa domain and the nuclease catalytic domain is located in the C-terminal 30 kDa domain. The purified RecBD1080ACD mutant is a processive helicase but not a nuclease, demonstrating that RecBCD has a single nuclease active site in the C-terminal 30 kDa domain of RecB.
RecD2 from Deinococcus radiodurans is a superfamily 1 DNA helicase that is homologous to the Escherichia coli RecD protein but functions outside the context of RecBCD enzyme. We report here on the ...kinetics of DNA unwinding by RecD2 under single and multiple turnover conditions. There is little unwinding of 20-bp substrates by preformed RecD2-dsDNA complexes when excess ssDNA is present to trap enzyme molecules not bound to the substrate. A shorter 12-bp substrate is unwound rapidly under single turnover conditions. The 12-bp unwinding reaction could be simulated with a mechanism in which the DNA is unwound in two kinetic steps with rate constant of kunw = 5.5 s−1 and a dissociation step from partially unwound DNA of koff = 1.9 s−1. These results indicate a kinetic step size of about 3–4 bp, unwinding rate of about 15–20 bp/s, and low processivity (p = 0.74). The reaction time courses with 20-bp substrates, determined under multiple turnover conditions, could be simulated with a four-step mechanism and rate constant values very similar to those for the 12-bp substrate. The results indicate that the faster unwinding of a DNA substrate with a forked end versus only a 5′-terminal single-stranded extension can be accounted for by a difference in the rate of enzyme binding to the DNA substrates. Analysis of reactions done with different RecD2 concentrations indicates that the enzyme forms an inactive dimer or other oligomer at high enzyme concentrations. RecD2 oligomers can be detected by glutaraldehyde cross-linking but not by size exclusion chromatography.
The RecB subunit of the Escherichia coli RecBCD enzyme has both helicase and nuclease activities. The helicase function was localized to an N-terminal domain, whereas the nuclease activity was found ...in a C-terminal domain. Recent analysis has uncovered a group of proteins that have weak amino acid sequence similarity to the RecB nuclease domain and that are proposed to constitute a family of related proteins (Aravind, L., Walker, D. R., and Koonin, E. V. (1999) Nucleic Acids Res. 27, 1223–1242). One is the E. coli RecE protein (exonuclease VIII), an ATP-independent exonuclease that degrades the 5′-terminated strand of double-stranded DNA. We have made mutations in several residues of RecE that align with the critical residues of RecB, and we find that the mutations reduce or abolish the nuclease activity of RecE but do not affect the enzyme binding to linear double-stranded DNA. Proteolysis experiments with subtilisin show that a stable 34-kilodalton C-terminal domain that contains these critical residues has nuclease activity, whereas no stable proteolytic fragments accumulate from the N-terminal portion of RecE. These results show that RecE has a nuclease domain and active site that are similar to RecB, despite the very weak sequence similarity between the two proteins. These similarities support the hypothesis that the nuclease domains of the two proteins are evolutionarily related.
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.
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 RecBN, 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 RecBN protein in which an aspartate residue has been changed to alanine. Structural analysis of the wild-type and mutant RecBN proteins by second derivative absorbance and circular dichroism spectro-scopy 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.
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 is an ATP-dependent nuclease on both single-stranded and double-stranded DNA substrates. We have investigated the kinetics of the RecBCD-catalyzed reaction with small, ...single-stranded oligodeoxyribonucleotide substrates under single-turnover conditions using rapid-quench flow techniques. RecBCD–DNA complexes were allowed to form in pre-incubation mixtures. The nuclease reactions were initiated by mixing with ATP. The reaction time-courses were fit to several possible reaction mechanisms and quantitative estimates were obtained for rate constants for individual reaction steps. The relative rates of forward reaction
versus dissociation from the DNA, and the fact that inclusion of excess non-radiolabeled single-stranded DNA to trap free RecBCD has no effect on the nuclease reaction, indicates that the reaction is processive. The reaction products show that the reaction begins near the 3′-end of the 5′-
32PDNA substrates and the major cleavage sites are two to four phosphodiester bonds apart. The product distribution is unchanged as the ATP concentration varies from 10 μM to 100 μM ATP, while the overall reaction rate varies by about tenfold. These observations suggest that DNA cleavage is tightly coordinated with movement of the enzyme along the DNA. The reaction time-courses at low concentrations of ATP (10 μM and 25 μM) have a significant lag before cleavage products appear. We propose that the lag represents ATP-dependent movement of the DNA from an initial binding site in the helicase domain of the RecB subunit to the nuclease active site in a separate domain of RecB. The extent of reaction of the substrate is limited (approximately 50%) under all conditions. This may indicate the formation of a non-productive RecBCD–DNA complex that does not dissociate in the 1–2 s time-scale of our experiments.
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.
We have expressed the RecD subunit of the RecBCD enzyme from Escherichia coli as a fusion protein with a 31-amino acid NH 2 -terminal extension including 6 consecutive histidine residues (HisRecD). ...The overexpressed fusion protein can be purified
in urea-denatured form by metal chelate affinity chromatography. The mixture of renatured HisRecD protein and the RecB and
RecC proteins has a high level of ATP-dependent nuclease activity with either single- or double-stranded DNA, enhanced DNA
unwinding activity, enhanced ATP hydrolysis activity in the presence of a small DNA oligomer cosubstrate, and Ï-cutting activity.
These are all characteristics of the RecBCD holoenzyme. The HisRecD protein by itself hydrolyzes ATP in the presence of high
concentrations of single-stranded DNA (polydeoxythymidine). The activity is unstable at 37°C, but is measurable at room temperature
(about 23°C). The HisRecD has very little ATPase activity in the presence of a much shorter single-stranded DNA (oligodeoxy(thymidine) 12 ). HisRecD hydrolyzes ATP more efficiently than GTP and UTP, and has very little activity with CTP. We also purified a fusion
protein containing a Lys to Gln mutation in the putative ATP-binding site of RecD. This mutant protein has no ATPase activity,
indicating that the observed ATP hydrolysis activity is intrinsic to the RecD protein itself.
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