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At least 19 recordsLinked to original sources

PubMed1992

Recombination and UV resistance of Escherichia coli with the cloned recA and recBCD genes of Serratia marcescens and Proteus mirabilis: evidence for an advantage of intraspecies combination of P. mirabilis RecA protein and RecBCD enzyme.

In Escherichia coli, constituents of the main recombination pathway are provided by the genes recA (RecA protein) and recBCD (RecBCD enzyme). Recombination in conjugation experiments and repair of UV damage of E. coli mutants deleted for recA, for recBCD or for recA plus recBCD were restored, although to different degrees, by the cloned recA and recBCD genes from Serratia marcescens or Proteus mirabilis. When both recombination enzymes were from the same species, repair and recombination efficiencies had the order E. coli greater than S. marcescens greater than P. mirabilis. However, the P. mirabilis recA plus recBCD genes resulted in higher levels of repair and recombination than those obtained with one component from P. mirabilis (recA or recBCD) and the other from E. coli or S. marcescens. The data provide evidence for the similarity of RecABCD pathways of recombination among enteric bacteria and suggest an in vivo advantage of an intraspecies combination of P. mirabilis RecA protein and RecBCD enzyme over interspecies combinations. This could point to a cooperation between these basic recombination enzymes. The molecular processes which could be involved are discussed.

Cloning, Molecular↗

PubMed1994

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.

Bacteriophage P22↗

PubMed1989

Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay.

We describe an assay to measure the extent of enzymatic unwinding of DNA by a DNA helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the DNA unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consistent with the known properties of recBCD enzyme, and closely parallels other methods for measuring recBCD enzyme helicase activity. The effects of varying temperature, substrate concentrations, enzyme concentration, and mono- and divalent salt concentrations on the helicase activity of recBCD enzyme were characterized. The apparent Km values for recBCD enzyme helicase activity on linear M13 dsDNA molecules at 25 degrees C are 0.6 nM dsDNA molecules and 130 microM ATP, respectively. The apparent turnover number for unwinding is approximately 15 microM base pairs s-1 (microM recBCD enzyme)-1. When this rate is corrected for the observed stoichiometry of recBCD enzyme binding to dsDNA, kcat for helicase activity corresponds to an unwinding rate of approximately 250 base pairs of DNA s-1 (functional recBCD complex)-1 at 25 degrees C. At 37 degrees C, the apparent Km value for dsDNA molecules was the same as that at 25 degrees C, but the apparent turnover number became 56 microM base pairs s-1 (microM recBCD enzyme)-1 [or 930 base pairs s-1 (functional recBCD complex)-1 when corrected for observed stoichiometry]. With increasing NaCl concentration, kcat peaks at 100 mM, and the apparent Km value for dsDNA increases by 3-fold at 200 mM NaCl. In the presence of 5 mM calcium acetate, the apparent Km value is increased by 3-fold, and kcat decreased by 20-30%. We have also shown that recBCD enzyme molecules are able to catalytically unwind additional dsDNA substrates subsequent to initiation, unwinding, and dissociation from a previous dsDNA molecule.

Acetates↗

PubMed2000

Bacteriophage P22 Abc2 protein binds to RecC increases the 5' strand nicking activity of RecBCD and together with lambda bet, promotes Chi-independent recombination.

Bacteriophage P22 Abc2 protein binds to the RecBCD enzyme from Escherichia coli to promote phage growth and recombination. Overproduction of the RecC subunit in vivo, but not RecB or RecD, interfered with Abc2-induced UV sensitization, revealing that RecC is the target for Abc2 in vivo. UV-induced ATP crosslinking experiments revealed that Abc2 protein does not interfere with the binding of ATP to either the RecB or RecD subunits in the absence of DNA, though it partially inhibits RecBCD ATPase activity. Productive growth of phage P22 in wild-type Salmonella typhimurium correlates with the presence of Abc2, but is independent of the absolute level of ATP-dependent nuclease activity, suggesting a qualitative change in the nature of Abc2-modified RecBCD nuclease activity relative to the native enzyme. In lambda phage crosses, Abc2-modified RecBCD could substitute for lambda exonuclease in Red-promoted recombination; lambda Gam could not. In exonuclease assays designed to examine the polarity of digestion, Abc2 protein qualitatively changes the nature of RecBCD double-stranded DNA exonuclease by increasing the rate of digestion of the 5' strand. In this respect, Abc2-modified RecBCD resembles a RecBCD molecule that has encountered the recombination hotspot Chi. However, unlike Chi-modified RecBCD, Abc2-modified RecBCD still possesses 3' exonuclease activity. These results are discussed in terms of a model in which Abc2 converts the RecBCD exonuclease for use in the P22 phage recombination pathway. This mechanism of P22-mediated recombination distinguishes it from phage lambda recombination, in which the phage recombination system (Red) and its anti-RecBCD function (Gam) work independently.

Adenosine Triphosphatases↗

PubMed1991

Interaction of RecBCD enzyme with DNA damaged by gamma radiation.

The DNA of a gene 2 mutant (T4 2-) of phage T4 is degraded by RecBCD enzyme in the bacterial cytoplasm. Under normal conditions, recBCD+ cells are therefore incapable of supporting the growth of phage T4 2-. Only if the nucleolytic activity of RecBCD enzyme is absent from the cytoplasm are T4 2(-)-infected bacteria able to form plaques. We found that recBCD+ cells can form plaques if, before infection with T4 2-, they have been exposed to gamma radiation. It is suggested that gamma ray-induced lesions of the bacterial DNA (e.g., double-strand breaks) bind RecBCD enzyme. This binding enables the enzyme to begin to degrade the bacterial chromosome, but simultaneously prevents its degradative action on the ends of minor DNA species, such as unprotected infecting phage chromosomes. Degradation of the chromosomal DNA, which occurs during the early postirradiation period, ceases about 60 min after gamma ray exposure. The reappearance of the nucleolytic action of RecBCD enzyme on T4 2- DNA accompanies the cessation of degradation of bacterial DNA. Both, this cessation and the reappearance of the nucleolytic action of ReCBCD enzyme on T4 2- DNA depend on a functional recA gene product. These results suggest that postirradiation DNA degradation is controlled by the recA-dependent removal of RecBCD enzyme from the damaged chromosome. By making use of the temperature-sensitive mutant recB270, we showed that RecBCD-mediated repair of gamma ray-induced lesions occurs during the early postirradiation period, i.e. during postirradiation DNA degradation. It is shown that the RecD subunit of RecBCD enzyme also participates in this repair.

Cell Survival↗

PubMed1991

The recA-recBCD dependent recombination pathways of Serratia marcescens and Proteus mirabilis in Escherichia coli: functions of hybrid enzymes and hybrid pathways.

The physical maps of cloned recBCD gene regions of Serratia marcescens and Proteus mirabilis were correlated to genes located in this region. The genes thyA, recC, recB, recD and argA were organized as in Escherichia coli. The 3 rec genes code for the 3 different subunits of the RecBCD enzyme and produced enzymes promoting recombination and repair of UV damage in E coli. The recBCD-dependent stimulation of recombination at specific nucleotide sequences called Chi (Chi-activation) was determined in lambda red-gam-crosses. Chi-activation by the different RecBCD enzymes decreased in the order E coli greater than S marcescens greater than P mirabilis. In E coli cloned subunits genes from S marcescens and P mirabilis led to the formation of functional hybrid enzymes consisting of subunits from 2 or even 3 species. The origin of the RecC subunit present in the hybrid enzymes affected the degree of Chi-activation. Further, changes in Chi-activation occurred when the RecD subunit in the enzyme from E coli was replaced by RecD proteins from S marcescens or P mirabilis. This suggested that the RecD subunit determines not only whether or not Chi-activation is possible but also to which extent it occurs. Finally we have reconstituted recombination pathways of S marcescens and P mirabilis by combining the cloned recA and recBCD genes from these species in E coli deleted for recA and recBCD. Both pathways can efficiently promote recombination and repair. Studies are summarized which showed that levels of repair and recombination promoted by the recA-recBCD genes are mostly higher when the recA and recBCD genes came from the same species than from 2 different species (hybrid RecBCD recombination pathway). The data are interpreted to provide evidence that in vivo the RecA protein co-operates with the RecBCD enzyme in recombination and repair of UV damage.

Chromosome Mapping↗

PubMed1991

A combination of RNase H (rnh) and recBCD or sbcB mutations in Escherichia coli K12 adversely affects growth.

Colony forming ability of Escherichia coli strains carrying the rnh-339::cat mutant allele is strongly dependent on the recBCD and sbcB genes. A mutation inactivating either the RecBCD nuclease or exonuclease I (sbcB) is sufficient to restrict severely the efficiency of plating of strains carrying the rnh-339::cat mutation. Combining a non-lethal temperature-sensitive mutation in the RecBCD nuclease, recB270 (Ts) or recC271 (Ts), with rnh-339::cat renders strains temperature sensitive for growth, even though rnh+ strains with the recB270 (Ts) or recC271 (Ts) alleles are viable at 42 degrees C. The recombinational functions of the RecBCD nuclease can be excluded as the source of lethality on the basis of the following observations. Introduction of a recombination proficient, exonuclease defective recD1009 allele or production of the phage lambda GamS protein (an inhibitor of the RecBCD exonuclease activity) in an rnh-339::cat strain dramatically delays or impairs the ability of such strains to form colonies. Restoration of recombination proficiency by inclusion of an sbcB15 mutation with recB21 recC22 mutations does not restore the ability of the rnh-339::cat mutant strains to plate normally. A recBCD+ strain bearing the rnh-339::cat and sbcB15 mutations forms very few visible colonies after 24 h but forms colonies at normal frequencies after 48 h of incubation. Finally, plating efficiencies of strains are unaffected when the RecBCD recombination pathway is inactivated by introduction of recA56 into an rnh-339::cat strain. These results imply that the defective growth of rnh-339::cat recBCD strains is due to a defect in repair and not recombination mediated by either the RecBCD or the RecF pathway.

Alleles↗

PubMed2005

Gamma-irradiated RecD overproducers become permanent recB-/C- phenocopies for extrachromosomal DNA processing due to prolonged titration of RecBCD enzyme on damaged Escherichia coli chromosome.

The RecBCD enzyme of Escherichia coli consists of three subunits RecB, RecC and RecD. RecBCD enzyme activities are regulated by its interaction with recombination hotspot Chi. Biochemical and genetic evidence suggest that interaction with Chi affects RecD subunit, and that RecD polypeptide overproduction antagonizes this interaction, suggesting that intact RecD replaces a Chi-modified one. We used bacteria with fragmented chromosomes due to double-strand breaks inflicted by UV and gamma-irradiation to explore in which way increased concentrations of RecBCD's individual subunits affect DNA metabolism. We confirmed that RecD overproduction alters RecBCD-dependent DNA repair and degradation in E. coli. Also, we found that RecB and RecC overproduction did not affect these processes. To determine the basis for the effects of RecD polypeptide overproduction, we monitored activities of RecBCD enzyme on gamma-damaged chromosomal DNA and, in parallel, on lambda and T4 2 phage DNA duplexes provided at intervals. We found that gamma-irradiated wild-type bacteria became transient, and RecD overproducers permanent recB(-)/C(-) phenocopies for processing phage DNA that is provided in parallel. Since this inability of irradiated bacteria to process extrachromosomal DNA substrates coincided in both cases with ongoing degradation of chromosomal DNA, which lasted much longer in RecD overproducers, we were led to conclude that the RecB(-)/C(-) phenotype is acquired as a consequence of RecBCD enzyme titration on damaged chromosomal DNA. This conclusion was corroborated by our observation that no inhibition of RecBCD activity occurs in gamma-irradiated RecBCD overproducers. Together, these results strongly indicate that RecD overproduction prevents dissociation of RecBCD enzyme from DNA substrate and thus increases its processivity.

Bacteriophage T4↗

PubMed2004

RecBCD enzyme overproduction impairs DNA repair and homologous recombination in Escherichia coli.

The Escherichia coli RecBCD enzyme is a powerful helicase and nuclease that processes DNA molecules containing blunt double-strand DNA end. Mutants deprived of RecBCD enzyme functions are extremely sensitive to DNA-damaging agents, poorly viable and severely deficient in homologous recombination. Remarkably, such important cellular functions rely on only about 10 molecules of RecBCD present in a cell. To determine the effect of an increased concentration of RecBCD enzyme and its derivatives on cellular processes that depend on the enzyme, we introduced wild-type and mutant alleles of recBCD genes on a low-copy-number plasmid into recB and wild-type bacteria and assessed their capacity for DNA repair and homologous recombination. We found that the overproduction of RecBCD enzyme, as well as RecBC and their nuclease-deficient derivatives, impairs both DNA repair and homologous recombination in E. coli. We also show that chromosomal degradation was increased in gamma-irradiated bacteria overproducing RecBCD but not in those overproducing RecBC enzyme, indicating that the increased nuclease activity is not the reason for defective DNA repair and homologous recombination observed in those cells. Our collective results suggest that DNA binding and processive helicase activities of the overproduced RecBCD enzyme, or its derivates, impair DNA repair and homologous recombination in E. coli. The cells control these activities of RecBCD by maintaining its extremely low concentration, thereby allowing efficient DNA repair and homologous recombination.

Conjugation, Genetic↗

PubMed1996

Interactions of the RecBCD enzyme from Escherichia coli and its subunits with DNA, elucidated from the kinetics of ATP and DNA hydrolysis with oligothymidine substrates.

Oligothymidines eight nucleotides or longer stimulate ATP hydrolysis by the RecBC and RecBCD enzymes, and they are substrates for the ATP-stimulated nuclease activity of RecBCD. The steady-state kinetics of ATP hydrolysis by the RecBC enzyme are consistent with a single ATPase and DNA binding site. Results with RecBCD and RecBCD-K177Q [an enzyme with a Lys-to-Gln mutation in the ATP binding motif of the RecD subunit [Korangy, F., & Julin, D. A. (1992) J. Biol. Chem. 267, 1727-1732]] indicate that ATP hydrolysis by the RecB subunit is stimulated by pd(T)12 binding to a high-affinity site, while the RecD subunit hydrolyzes ATP stimulated by pd(T)12 binding to a low-affinity site. The site which stimulates RecB has about 50-fold greater affinity for DNA in either RecBCD or RecBCD-K177Q than does the corresponding site in RecBC. The rates of ATP hydrolysis observed for the RecBCD enzyme at low concentrations of pd(T)12 are best explained by a mechanism where the enzyme binds to the DNA and catalyzes multiple rounds of ATP hydrolysis before dissociating. Larger DNA molecules [pd(T)25-30 and poly(dT)] are bound more tightly by RecBCD, are hydrolyzed more rapidly, and are much more effective in stimulating ATP hydrolysis than is pd(T)12. The results at low ATP concentrations where the nuclease activity is minimal (5 microM) suggest that ATP hydrolysis is stimulated by the DNA ends, but there is no evidence that the RecBCD enzyme moves along these DNA molecules in an ATP-dependent manner under these conditions.

Adenosine Triphosphatases↗

PubMed1997

Chi-activated RecBCD enzyme possesses 5'-->3' nucleolytic activity, but RecBC enzyme does not: evidence suggesting that the alteration induced by Chi is not simply ejection of the RecD subunit.

BACKGROUND: Homologous recombination in Escherichia coli is initiated by the RecBCD enzyme, and is stimulated by DNA elements known as Chi (chi) sites. The RecBCD enzyme is both a helicase and a nuclease. Recognition of chi causes both attenuation of the 3'-->5' exonuclease activity of the RecBCD enzyme, and activation of an exonuclease activity with 5'-->3' polarity, while leaving the helicase activity unaffected. A variety of evidence suggests that chi-recognition by RecBCD enzyme is accompanied by ejection of the RecD subunit. RESULTS: Through examination of RecBCD exonuclease activity under a variety of conditions, we have shown that recognition of chi by the RecBCD enzyme results in a net reduction of nuclease activity. In addition, the exact location of the first cleavage event elicited by chi-activation of the 5'-->3' nuclease is dependent upon the concentration of free magnesium ions. Finally, we have demonstrated that purified RecBC enzyme (i.e. without the RecD subunit) possesses no significant exonuclease activity under conditions where the chi-modified RecBCD enzyme is an active 5'-->3' exonuclease. CONCLUSIONS: We have shown that, despite the activation of a 5'-->3' exonuclease, recognition of chi by the RecBCD enzyme results in a net preservation of DNA. This new chi-activated nucleolytic action shows surprising variability in the exact location of its initial cleavage. We have demonstrated that purified RecBC enzyme is not an exact analogue of the chi-activated RecBCD enzyme, suggesting that the biochemical basis of chi-activation is not simply ejection of the RecD subunit.

DNA↗

PubMed1994

Reversible inactivation of the Escherichia coli RecBCD enzyme by the recombination hotspot chi in vitro: evidence for functional inactivation or loss of the RecD subunit.

Genetic recombination in Escherichia coli is stimulated by a RecBCD enzyme-mediated event at DNA sequences known as Chi (chi) sites (5'-GCTGGTGG-3'). Previously, it was shown that chi acts to regulate the nuclease activity of RecBCD; here, we demonstrate that, under appropriate conditions, interaction with chi sites can also result in an inactivation of helicase activity of RecBCD. The unwinding of double-stranded DNA-containing chi sites, under conditions of limiting Mg2+ ion, results in the reversible inactivation of RecBCD; addition of excess Mg2+ to the reaction reactivates all activities of RecBCD. Inactivation is the consequence of a chi-dependent modification of RecBCD that appears to result from an inability of the chi-modified RecBCD to reinitiate unwinding of intact DNA molecules. This characteristic behavior of RecBCD and chi is displayed by the reconstituted RecBC (i.e., without the RecD subunit), except that it is not dependent on chi interaction. This biochemical similarity between the chi-modified RecBCD and RecBC enzymes implies that recognition of chi results in a dissociation or functional inactivation of RecD subunit and lends support to the hypothesis that interaction with chi results in ejection of the RecD subunit.

DNA Helicases↗

PubMed2006

The nuclease domain of the Escherichia coli RecBCD enzyme catalyzes degradation of linear and circular single-stranded and double-stranded DNA.

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.

Amino Acid Sequence↗

PubMed1992

Chi sequence protects against RecBCD degradation of DNA in vivo.

RecBCD is a multifunctional enzyme involved in DNA degradation and homologous recombination. It also produces an endonucleolytic cleavage near properly oriented chi sites (5'-GCTGGTGG-3'). Plasmids are not known to be affected by either RecBCD enzyme or the presence of a chi site. We report here that plasmids that replicate by a rolling circle mechanism accumulate large amounts of high molecular weight linear multimers (HMW), either if they contain a chi site or if RecBCD is absent. An in vivo inducible system for rolling circle replication was constructed to study RecBCD and its interactions with chi. Results show that (i) HMW accumulation is chi orientation dependent, and (ii) a succession of chi sites prevents degradation of HMW by RecBCD enzyme. These results demonstrate chi activity in plasmids. The rolling circle mechanism produces a sigma structure during plasmid replication; we propose that the double-stranded DNA tail of this sigma form allows RecBCD entry; the tail is degraded unless it is protected by a chi site. By analogy, a principal role of chi in the survival of lambda red-gam- mutants in wild-type strains may be to protect rolling circle concatemers (in late replication) from degradation by RecBCD.

Base Sequence↗

PubMed2004

Specific inhibition of the E.coli RecBCD enzyme by Chi sequences in single-stranded oligodeoxyribonucleotides.

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.

Base Sequence↗

PubMed1998

SSB protein controls RecBCD enzyme nuclease activity during unwinding: a new role for looped intermediates.

The RecBCD enzyme of Escherichia coli initiates homologous recombination by unwinding and simultaneously degrading DNA from a double-stranded DNA end. Single-stranded DNA loops are intermediates of this unwinding process. Here we show that SSB protein reduces the level of DNA degradation by RecBCD enzyme during unwinding, by binding to these ssDNA intermediates. Prior to interaction with the recombination hot spot chi, RecBCD enzyme has both 3'-->5' exonuclease and a weaker 5'-->3' exonuclease activity. We show that degradation of the 5'-terminal strand at the entry site is much more extensive in the absence of SSB protein. After interaction with chi, the level of 5'-->3' exonuclease activity is increased; as expected, degradation of the 5'-strand is also elevated in the absence of SSB protein. Furthermore, we show that, in the absence of SSB protein, the RecBCD enzyme is inhibited by the ssDNA products of unwinding; SSB protein alleviates this inhibition. These results provide insight into the organization of helicase and nuclease domains within the RecBCD enzyme, and also suggest a new level at which the nuclease activity of RecBCD enzyme is controlled. Hence, they offer new insight into the role of SSB protein in the initiation phase of recombination.

DNA Helicases↗

PubMed2005

Direct visualization of RecBCD movement reveals cotranslocation of the RecD motor after chi recognition.

In Escherichia coli, chi (5'-GCTGGTGG-3') is a recombination hotspot recognized by the RecBCD enzyme. Recognition of chi reduces both nuclease activity and translocation speed of RecBCD and activates RecA-loading ability. RecBCD has two motor subunits, RecB and RecD, which act simultaneously but independently. A longstanding hypothesis to explain the changes elicited by chi interaction has been "ejection" of the RecD motor from the holoenzyme at chi. To test this proposal, we visualized individual RecBCD molecules labeled via RecD with a fluorescent nanoparticle. We could directly see these labeled, single molecules of RecBCD moving at up to 1835 bp/s (approximately 0.6 microm/s). Those enzymes translocated to chi, paused, and continued at reduced velocity, without loss of RecD. We conclude that chi interaction induces a conformational change, resulting from binding of chi to RecC, and not from RecD ejection. This change is responsible for alteration of RecBCD function that persists for the duration of DNA translocation.

Biotinylation↗

PubMed2003

RecBCD enzyme is a bipolar DNA helicase.

Escherichia coli RecBCD is a heterotrimeric helicase/nuclease that catalyses a complex reaction in which double-strand breaks in DNA are processed for repair by homologous recombination. For some time it has been clear that the RecB subunit possesses a 3' --> 5' DNA helicase activity, which was thought to drive DNA translocation and unwinding in the RecBCD holoenzyme. Here we show that purified RecD protein is also a DNA helicase, but one that possesses a 5' --> 3' polarity. We also show that the RecB and RecD helicases are both active in intact RecBCD, because the enzyme remains capable of processive DNA unwinding when either of these subunits is inactivated by mutation. These findings point to a bipolar translocation model for RecBCD in which the two DNA helicases are complementary, travelling with opposite polarities, but in the same direction, on each strand of the antiparallel DNA duplex. This bipolar motor organization helps to explain various biochemical properties of RecBCD, notably its exceptionally high speed and processivity, and offers a mechanistic insight into aspects of RecBCD function.

Adenosine Triphosphatases↗