RESUMEN
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 k(unw) = 5.5 s(-1) and a dissociation step from partially unwound DNA of k(off) = 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.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Helicasas/química , Deinococcus/metabolismo , Exodesoxirribonucleasa V/química , Animales , Cromatografía/métodos , Reactivos de Enlaces Cruzados/química , ADN/química , ADN Helicasas/metabolismo , ADN de Cadena Simple/química , Dimerización , Relación Dosis-Respuesta a Droga , Exodesoxirribonucleasa V/metabolismo , Glutaral/química , Cinética , Modelos Biológicos , Oligonucleótidos/química , Desnaturalización ProteicaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Deinococcus/genética , Deinococcus/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , ADN/metabolismo , Reparación del ADN , Deinococcus/crecimiento & desarrollo , Deinococcus/efectos de la radiación , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Viabilidad Microbiana/efectos de la radiación , Mutación , Fenotipo , Pliegue de ProteínaRESUMEN
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.
Asunto(s)
Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , ADN Helicasas/genética , ADN Helicasas/metabolismo , Deinococcus/enzimología , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Antineoplásicos Alquilantes/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Southern Blotting , Clonación Molecular , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Deinococcus/genética , Rayos gamma , Peróxido de Hidrógeno/farmacología , Hidrólisis/efectos de los fármacos , Hidrólisis/efectos de la radiación , Técnicas In Vitro , Metilmetanosulfonato/farmacología , Mitomicina/farmacología , Datos de Secuencia Molecular , Mutación/genética , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Oxidantes/farmacología , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Rayos UltravioletaRESUMEN
The bacterium Deinococcus radiodurans is resistant to extremely high levels of DNA-damaging agents such as UV light, ionizing radiation, and chemicals such as hydrogen peroxide and mitomycin C. The organism is able to repair large numbers of double-strand breaks caused by ionizing radiation, in spite of the lack of the RecBCD enzyme, which is essential for double-strand DNA break repair in Escherichia coli and many other bacteria. The D. radiodurans genome sequence indicates that the organism lacks recB and recC genes, but there is a gene encoding a protein with significant similarity to the RecD protein of E. coli and other bacteria. We have generated D. radiodurans strains with a disruption or deletion of the recD gene. The recD mutants are more sensitive than wild-type cells to irradiation with gamma rays and UV light and to treatment with hydrogen peroxide, but they are not sensitive to treatment with mitomycin C and methyl methanesulfonate. The recD mutants also show greater efficiency of transformation by exogenous homologous DNA. These results are the first indication that the D. radiodurans RecD protein has a role in DNA damage repair and/or homologous recombination in the organism.
Asunto(s)
Proteínas Bacterianas/genética , Daño del ADN , Deinococcus/genética , Exodesoxirribonucleasa V/genética , Mutación , Proteínas Bacterianas/metabolismo , Southern Blotting , ADN Bacteriano/genética , Deinococcus/efectos de los fármacos , Deinococcus/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Exodesoxirribonucleasa V/metabolismo , Rayos gamma , Prueba de Complementación Genética , Peróxido de Hidrógeno/farmacología , Viabilidad Microbiana/efectos de los fármacos , Viabilidad Microbiana/genética , Viabilidad Microbiana/efectos de la radiación , Transformación Bacteriana , Rayos UltravioletaRESUMEN
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'-32P]DNA 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 microM to 100 microM 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 microM and 25 microM) 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.
Asunto(s)
Adenosina Trifosfato/farmacología , Escherichia coli/enzimología , Exodesoxirribonucleasa V/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Bases , ADN/genética , ADN/metabolismo , Cinética , Datos de Secuencia Molecular , Oligonucleótidos/metabolismo , Factores de TiempoRESUMEN
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.
Asunto(s)
ADN de Cadena Simple/metabolismo , ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonucleasa V/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Dominio Catalítico , Cationes Bivalentes , Electroforesis en Gel de Poliacrilamida , Proteínas de Escherichia coli/genética , Exodesoxirribonucleasa V/genética , Metales/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Mutación , Conformación de Ácido Nucleico , Factores de TiempoRESUMEN
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 5'-single-stranded tail or a forked end, but not blunt-ended or 3'-tailed duplexes. Duplexes with 10-12 nucleotide (nt) 5'-tails are good unwinding substrates and are bound tightly, while DNA with shorter tails (4-8 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 5'-tails). Unwinding of the longer substrates is stimulated somewhat (4-5-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 5'-3' polarity and low processivity.
Asunto(s)
ADN Helicasas/química , ADN Helicasas/metabolismo , Deinococcus/enzimología , Exodesoxirribonucleasa V/química , Exodesoxirribonucleasa V/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Bases , ADN Helicasas/genética , Reparación del ADN , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Deinococcus/genética , Exodesoxirribonucleasa V/genética , Genoma Bacteriano , Hidrólisis , Datos de Secuencia Molecular , Estructura Molecular , Especificidad por SustratoRESUMEN
RecBCD is an ATP-dependent helicase and exonuclease which generates 3' single-stranded DNA (ssDNA) ends used by RecA for homologous recombination. The exonuclease activity is altered when RecBCD encounters a Chi sequence (5'-GCTGGTGG-3') in double-stranded DNA (ds DNA), an event critical to the generation of the 3'-ssDNA. This study tests the effect of ssDNA oligonucleotides having a Chi sequence (Ch+) or a single base change that abolishes the Chi sequence (Chi(o)), on the enzymatic activities of RecBCD. Our results show that a 14 and a 20mer with Chi+ in the center of the molecule inhibit the exonuclease and helicase activities of RecBCD to a greater extent than the corresponding Chi(o) oligonucleotides. Oligonucleotides with the Chi sequence at one end, or the Chi sequence alone in an 8mer, failed to show Chi-specific inhibition of RecBCD. Thus, Chi recognition requires that Chi be flanked by DNA at either end. Further experiments indicated that the oligonucleotides inhibit RecBCD from binding to its dsDNA substrate. These results suggest that a specific site for Chi recognition exists on RecBCD, which binds Chi with greater affinity than a non-Chi sequence and is probably adjacent to non-specific DNA binding sites.