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 mechanism by which double-strand DNA breaks are repaired in the radiation-resistant bacterium
Deinococcus radiodurans is not well understood. This organism lacks the RecBCD helicase/nuclease, ...which processes broken DNA ends in other bacteria. The RecF pathway is an alternative pathway for recombination and DNA repair in
E. coli, when RecBCD is absent due to mutation, and
D. radiodurans may rely on enzymes of this pathway for double-strand break repair. The RecJ exonuclease is thought to process broken DNA ends for the RecF pathway. We attempted to delete the
recJ gene from
D. radiodurans, using homologous recombination to replace the gene with a streptomycin-resistance cassette. We were unable to obtain a complete deletion mutant, in which the gene is deleted from all of the chromosome copies in this polyploid organism. Quantitative real-time PCR shows that the heterozygous mutants have a
recJ gene copy that is
ca. 10–30% that of the wild-type. Mutants with reduced
recJ gene copy grow slowly and are more sensitive than wild-type to UV irradiation, gamma irradiation, and hydrogen peroxide. The mutants are as resistant as wild-type to methyl-methanesulfonate. The
D. radiodurans RecJ protein was expressed in
E. coli and purified under denaturing conditions. The re-folded protein has nuclease activity on single-stranded DNA with specificity similar to that of
E. coli RecJ exonuclease.
The bacterium Deinococcus radiodurans is extremely resistant to high levels of DNA-damaging agents, including gamma rays and ultraviolet light that can lead to
double-stranded DNA breaks. ...Surprisingly, the organism does not appear to have a RecBCD enzyme, an enzyme that is critical
for double-strand break repair in many other bacteria. The D. radiodurans genome does encode a protein whose closest characterized homologues are RecD subunits of RecBCD enzymes in other bacteria.
We have purified this novel D. radiodurans RecD protein and characterized its biochemical activities. The D. radiodurans RecD protein is a DNA helicase that unwinds short (20 base pairs) DNA duplexes with either a 5â²-single-stranded tail or a
forked end, but not blunt-ended or 3â²-tailed duplexes. Duplexes with 10â12 nucleotide (nt) 5â²-tails are good unwinding substrates
and are bound tightly, while DNA with shorter tails (4â8 nt) are poor unwinding substrates and are bound much less tightly.
The RecD protein is much less efficient at unwinding slightly longer substrates (52 or 76 base pairs, with 12 nt 5â²-tails).
Unwinding of the longer substrates is stimulated somewhat (4â5-fold) by the single-stranded DNA-binding protein from D. radiodurans . These results show that the D. radiodurans RecD protein is a DNA helicase with 5â²-3â² polarity and low processivity.
Deinococcus radiodurans survives extremely high doses of ionizing and ultraviolet radiation and treatment with various DNA-damaging chemicals. As an effort to identify and characterize proteins that ...function in DNA repair in this organism, we have studied the protein encoded by locus DR1572. This gene is predicted to encode a Superfamily I DNA helicase, except that genome sequencing indicated that it has a one-base frameshift and would not encode a complete helicase. We have cloned the gene from two different
D. radiodurans strains and find that the frameshift mutation is not present. The corrected gene encodes a 755 residue protein that is similar to the
Bacillus subtilis YvgS protein and to helicase IV of
Escherichia coli. The purified protein (helicase IV
Dr) has ATP hydrolysis and DNA helicase activity. A truncated protein that lacks 214 residues from the N-terminus, which precede the conserved helicase domain, has greater ATPase activity than the full-length protein but has no detectable helicase activity. Disruption of locus DR1572 in the
D. radiodurans chromosome causes greater sensitivity to hydrogen peroxide and methyl-methanesulfonate compared to wild-type cells, but no change in resistance to gamma and ultraviolet radiation and to mitomycin C. The results indicate that locus DR1572 encodes a complete protein that contributes to DNA metabolism in
D. radiodurans.
The RecBCD enzyme from Escherichia coli is an ATP-dependent helicase and an ATP-stimulated nuclease. The 3′→ 5′exonuclease activity on double-stranded DNA is suppressed when the enzyme encounters a ...recombinational hot spot, called chi (χ ). We have prepared a RecB deletion mutant (RecB1-929) by using results of limited proteolysis experiments that indicated that the RecB subunit consists of two main domains. The RecB1-929protein, comprising the 100-kDa N-terminal domain of RecB, is an ATP-dependent helicase and a single-stranded DNA-dependent ATPase. Reconstitution of RecB1-929with RecC and RecD leads to processive unwinding of a linearized plasmid. However, the reconstituted RecB1-929CD enzyme has lost the single-strand endo- and exonuclease and the double-strand exonuclease activities of the RecBCD enzyme. These results show that the 30-kDa C-terminal domain of RecB has an important role in the nuclease activity of RecBCD. On the basis of these findings, we propose the RecB C-terminal domain swing model to explain RecBCD's transformation from a 3′→ 5′exonuclease to a helicase when it meets a χ site.
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.
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 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.
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