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PubMed1987

Kinetics of recB-dependent repair: relationship to post-UV inactivation of the prophage.

By making use of the temperature-sensitive mutant recB270, we showed that the RecBCD enzyme is needed for repair between 1 and 4 h after UV exposure. recB-dependent prophage inactivation (Petranović et al. (1984), Mol. Gen. Genet., 196, 167-169) takes place in all dying cells during the same period of time. The kinetics of decrease in the yield of recombinants in phage-propage crosses resemble those of prophage inactivation in UV-irradiated bacteria. This indicates that recombination processes (including site-specific recombination required for prophage excision) are blocked in cells destined to die. On the basis of our results, we suggest that a large fraction of damaged cells is rescued by the RecA-RecBCD recombination pathway. If repair is unsuccessful, RecA-RecBCD recombination intermediates persist in the irradiated cells leading to prophage inactivation.

Coliphages↗
PubMed1987

recA-dependent DNA repair in UV-irradiated Escherichia coli.

UV-radiation-induced lesions in DNA result in the formation of excision gaps, daughter-strand gaps (DSG) and double-strand breaks (DSB), which are repaired by several different mechanisms. Postreplication repair. The recA gene is a master gene that controls all of the pathways of postreplication repair. The repair of DSG proceeds by one pathway that is also recF dependent, and one pathway that is constitutive and independent of the recF and recBC genes. A small fraction of the recF recB-independent repair of DSG is dependent upon the umuC gene, and may define an error-prone pathway of postreplication repair. Unrepaired DSG can be converted to DSB, which are normally repaired by the RecBCD pathway. However, in the recBC sbcB background, these DSB are repaired by a recF-dependent process. The RecF pathways of postreplication repair appear to utilize DNA containing a single-stranded region (either a gap or a DSB with a single-stranded end), while the RecBCD pathway appears to utilize the blunt ends of duplex DNA to promote the recombinational repair of DSB. The polA gene (especially the 5'----3' exonuclease activity of DNA polymerase I) functions in pathways of postreplication repair (both for the repair of DSG and DSB) that are largely independent of the recF gene. Nucleotide excision repair. The repair of excision gaps is independent of the recA gene in cells with unreplicated chromosomes, but is recA dependent in cells with partially replicated chromosomes at the time of UV irradiation. This recA-dependent repair of excision gaps appears to be analogous to the recF- and recB-dependent pathways of postreplication repair, i.e. the RecF pathway repairs DNA gaps, and the RecBCD pathway repairs the DSB that arise at unrepaired gaps.

DNA Repair↗
PubMed2005

Inactivation of recG stimulates the RecF pathway during lesion-induced recombination in E. coli.

Lesions that transiently block DNA synthesis generate replication intermediates with recombinogenic potential. In order to investigate the mechanisms involved in lesion-induced recombination, we developed an homologous recombination assay involving the transfer of genetic information from a plasmid donor molecule to the Escherichia coli chromosome. The replication blocking lesion used in the present assay is formed by covalent binding of the carcinogen N-2-acetylaminofluorene to the C8 position of guanine residues (G-AAF adducts). The frequency of recombination events was monitored as a function of the number of lesions present on the donor plasmid. These DNA adducts are found to trigger high levels of homologous recombination events in a dose-dependent manner. Formation of recombinants is entirely RecA-dependent, the RecF and RecBCD sub-pathways accounting for about 2/3 and 1/3, respectively. Inactivation of recG stimulates recombinant formation about five-fold. In a recG background, the RecF pathway is stimulated about four-fold, while the contribution of the RecBCD pathway remains constant. In addition, in the recG strain, a recombination pathway that accounts for about 30% of the recombinants and requires genes that belong to both RecF and RecBCD pathways is revealed.

2-Acetylaminofluorene↗
PubMed2005

The type I restriction endonuclease EcoR124I, couples ATP hydrolysis to bidirectional DNA translocation.

Type I restriction endonuclease holoenzymes contain methylase (M), restriction (R) and specificity (S) subunits, present in an M2:R2:S1 stoichiometry. These enzymes bind to specific DNA sequences and translocate dsDNA in an ATP-dependent manner toward the holoenzyme anchored at the recognition sequence. Once translocation is impeded, DNA restriction, which functions to protect the host cell from invading DNA, takes place. Translocation and DNA cleavage are afforded by the two diametrically opposed R-subunits. To gain insight into the mechanism of translocation, a detailed characterization of the ATPase activity of EcoR124I was done. Results show that following recognition sequence binding, ATP hydrolysis-coupled, bidirectional DNA translocation by EcoR124I ensues, with the R-subunits transiently disengaging, on average, every 515 bp. Macroscopic processivity of 2031(+/-184)bp is maintained, as the R-subunits remain in close proximity to the DNA through association with the methyltransferase. Transient uncoupling of ATP hydrolysis from translocation results in 3.1(+/-0.4) ATP molecules being hydrolyzed per base-pair translocated per R-subunit. This is the first clear demonstration of the coupling of ATP hydrolysis to dsDNA translocation, albeit inefficient. Once translocation is impeded on supercoiled DNA, the DNA is cleaved. DNA cleavage inactivates the EcoR124I holoenzyme partially and reversibly, which explains the stoichiometric behaviour of type I restriction enzymes. Inactivated holoenzyme remains bound to the DNA at the recognition sequence and immediately releases the nascent ends. The release of nascent ends was demonstrated using a novel, fluorescence-based, real-time assay that takes advantage of the ability of the Escherichia coli RecBCD enzyme to unwind restricted dsDNA. The resulting unwinding of EcoR124I-restricted DNA by RecBCD reveals coordination between the restriction-modification and recombination systems that functions to destroy invading DNA efficiently. In addition, we demonstrate the displacement of EcoR124I following DNA cleavage by the translocating RecBCD enzyme, resulting in the restoration of catalytic function to EcoR124I.

Adenosine Triphosphatases↗
PubMed2000

PCR-mediated gene replacement in Escherichia coli.

The hyper-recombinogenic properties of an E. coli strain in which the recBCD genes have been replaced by lambda red recombination functions were exploited in the development of a general PCR-mediated gene replacement scheme for Escherichia coli. Linear DNA substrates generated by recombinant PCR are introduced by electroporation into strains containing the recBCDDelta::red substitution. This technique allows for gene replacement in E. coli without prior cloning of the gene of interest. In addition, the counter-selectable marker sacB has been used to construct unmarked precise gene deletions without the need to form sacB-containing plasmid integrates. In other experiments, electroporation of recBCDDelta::red strains with high concentrations of linear DNA fragments (derived from plasmid digests) gave linear transformation rates approaching 1% of the survivors of electroporation. The placement of lambda red and gam at a locus in the chromosome other than recBCD (galK) resulted in a strain that was as hyper-rec as one containing the lambda red for recBCD substitution. The gene replacement technique described here has been used for the construction of deletion-substitution alleles of lacZ and sulA, as well as six genes important for general homologous recombination in E. coli. Three of these replacements were performed without prior cloning of the genes.

Bacteriophage lambda↗
PubMed2000

Orientation specificity of the Lactococcus lactis Chi site.

BACKGROUND: In Escherichia coli, the Chi sequence modulates the activity of RecBCD, a powerful double-stranded (ds) DNA exonuclease/helicase. Chi attenuates RecBCD exonuclease activity and stimulates homologous recombination in an orientation-dependent manner. ChiEc is frequent and over-represented on its genome, which is thought to be related to its role in dsDNA break repair. We previously identified a Chi-like sequence (referred to as ChiLl) and an exonuclease/helicase in the Gram-positive bacterium Lactococcus lactis. ChiLl and RexAB are functional analogues of ChiEc and RecBCD. RESULTS: We report that ChiLl attenuates RexAB exonuclease activity and stimulates homologous recombination in an orientation-dependent manner. Analysis of ChiLl distribution on the L. lactis chromosome reveals that ChiLl is frequent, highly over-represented, and oriented with respect to the direction of replication. CONCLUSION: Our results show that a single orientation of ChiLl interacts with RexAB. The active orientation is preferentially found on the replication leading strand of the L. lactis genome, consistent with a primary role of ChiLl in repair of dsDNA breaks at the replication fork. We propose that orientation-dependence of Chi activity and over-representation of Chi sequences on bacterial genomes may be conserved properties of exonuclease/helicase-Chi couples. Other properties of the Chi sequence distribution on the genomes might reflect more specific characteristics of each couple and of the host.

Chromosome Inversion↗
PubMed1998

Identification of the lactococcal exonuclease/recombinase and its modulation by the putative Chi sequence.

Studies of RecBCD-Chi interactions in Escherichia coli have served as a model to understand recombination events in bacteria. However, the existence of similar interactions has not been demonstrated in bacteria unrelated to E. coli. We developed an in vivo model to examine components of dsDNA break repair in various microorganisms. Here, we identify the major exonuclease in Lactococcus lactis, a Gram-positive organism evolutionarily distant from E. coli, and provide evidence for exonuclease-Chi interactions. Insertional mutants of L. lactis, screened as exonuclease-deficient, affected a single locus and resulted in UV sensitivity and recombination deficiency. The cloned lactococcal genes (called rexAB) restored UV resistance, recombination proficiency, and the capacity to degrade linear DNA, to an E. coli recBCD mutant. In this context, DNA degradation is specifically blocked by the putative lactococcal Chi site (5'-GCGCGTG-3'), but not by the E. coli Chi (5'-GCTGGTGG-3') site. RexAB-mediated recombination was shown to be stimulated approximately 27-fold by lactococcal Chi. Our results reveal that RexAB fulfills the biological roles of RecBCD and indicate that its activity is modulated by a short DNA sequence. We speculate that exonuclease/recombinase enzymes whose activities are modulated by short DNA sequences are widespread among bacteria.

Base Sequence↗
PubMed1998

The reduced levels of chi recognition exhibited by the RecBC1004D enzyme reflect its recombination defect in vivo.

Homologous recombination in Escherichia coli is initiated by the RecBCD enzyme and is stimulated by an 8-nucleotide element known as Chi (chi). We present a detailed biochemical characterization of a mutant RecBCD enzyme, designated RecBC1004D, that displays a reduced level of chi site recognition. Initially characterized genetically as unable to respond to the chi sequence, we provide evidence to indicate that the ability of this mutant enzyme to respond to chi is reduced rather than lost; the mutant displays about 20-fold lower chi recognition than wild-type RecBCD enzyme. Although this enzyme exhibits wild-type levels of double-stranded DNA exonuclease, helicase, and ATPase activity, its ability to degrade single-stranded DNA is enhanced 2-3-fold. The data presented here suggest that the reduced recombination proficiency of the recBC1004D strain observed in vivo results from a basal level of modification of the RecBC1004D enzyme at both chi-specific, as well as nonspecific, DNA sequences.

Adenosine Triphosphatases↗
PubMed1997

Clustering of chi sequence in Escherichia coli genome.

An 8-mer DNA sequence called chi (5'-GCTGGTGG) is present on the Escherichia coli chromosome at a high frequency. It is responsible for both the attenuation of RecBCD exonuclease activity and the promotion of RecABCD-mediated homologous recombination. chi was first identified as a site that increased plaque size of bacteriophage lambda. lambda containing chi makes very small plaques on a recC* (recC1004) mutant because chi is poorly recognized by the RecBC*D mutant enzyme. We cloned E. coli chromosomal fragments in lambda that allowed lambda to form larger plaques on this recC* mutant as well as on the rec+ parent. One identified fragment contained a cluster of two copies of chi and several chi-like sequences with the same orientation. It increased recombination in the rec+ strain more than a fragment with one chi did. This fragment was within the rep gene, whose helicase product is known to be required for growth in the absence of functional RecBCD enzyme. The possibility that RecBCD enzyme might interact both with the rep gene and its product is discussed. Many of the other chi clusters identified in the E. coli genome database lie within genes for membrane proteins. The possible significance of these findings is discussed.

Bacteriophage lambda↗
PubMed1988

The genetic dependence of recombination in recD mutants of Escherichia coli.

RecBCD enzyme has multiple activities including helicase, exonuclease and endonuclease activities. Mutations in the genes recB or recC, encoding two subunits of the enzyme, reduce the frequency of many types of recombinational events. Mutations in recD, encoding the third subunit, do not reduce recombination even though most of the activities of the RecBCD enzyme are severely reduced. In this study, the genetic dependence of different types of recombination in recD mutants has been investigated. The effects of mutations in genes in the RecBCD pathway (recA and recC) as well as the genes specific for the RecF pathway (recF, recJ, recN, recO, recQ, ruv and lexA) were tested on conjugational, transductional and plasmid recombination, and on UV survival. recD mutants were hyper-recombinogenic for all the monitored recombination events, especially those involving plasmids, and all recombination events in recD strains required recA and recC. In addition, unlike recD+ strains, chromosomal recombination events and the repair of UV damage to DNA in recD strains were dependent on one RecF pathway gene, recJ. Only a subset of the tested recombination events were affected by ruv, recN, recQ, recO and lexA mutations.

Chromosomes, Bacterial↗
PubMed1988

Chain-bias of Escherichia coli Rec-mediated lambda patch recombinants is independent of the orientation of lambda cos.

Chi is a hotspot for homologous recombination mediated by the RecBCD (Rec) pathway of Escherichia coli. For Rec-mediated recombination of phage lambda, the orientation of lambda cos in the lambda chromosome dictates the direction of travel of RecBCD enzyme through DNA and dictates which orientation of Chi or Chi-like sequences will be active in stimulating recombination. I previously found that Rec-mediated lambda patch heteroduplexes, stimulated by Chi or not, are chain-biased; at the lambda P locus, recombinant information resides on the lambda r chain. This bias exists in the presence or absence of Chi sites. Reported herein is the finding that r-chain-bias at the P locus is independent of the orientation of lambda cos and thus also independent of the orientation of active Chi's or Chi-like sequences and of the direction of travel of RecBCD enzyme. These results disprove previously elaborated models in which a chain-specific nick at Chi initiates recombination, and imply that some other chain-distinguishing process is involved with recombination. Replication and transcription are candidates for such a process.

Bacteriophage lambda↗
PubMed1998

Chi-dependent intramolecular recombination in Escherichia coli.

Homologous recombination in Escherichia coli is enhanced by a cis-acting octamer sequence named Chi (5'-GCTGGTGG-3') that interacts with RecBCD. To gain insight into the mechanism of Chi-enhanced recombination, we recruited an experimental system that permits physical monitoring of intramolecular recombination by linear substrates released by in vivo restriction from infecting chimera phage. Recombination of the released substrates depended on recA, recBCD and cis-acting Chi octamers. Recombination proficiency was lowered by a xonA mutation and by mutations that inactivated the RuvABC and RecG resolution enzymes. Activity of Chi sites was influenced by their locations and by the number of Chi octamers at each site. A single Chi site stimulated recombination, but a combination of Chi sites on the two homologs was synergistic. These data suggest a role for Chi at both ends of the linear substrate. Chi was lost in all recombinational exchanges stimulated by a single Chi site. Exchanges in substrates with Chi sites on both homologs occurred in the interval between the sites as well as in the flanking interval. These observations suggest that the generation of circular products by intramolecular recombination involves Chi-dependent processing of one end by RecBCD and pairing of the processed end with its duplex homolog.

Bacteriophages↗
PubMed1999

The RecBC enzyme loads RecA protein onto ssDNA asymmetrically and independently of chi, resulting in constitutive recombination activation.

Double-strand DNA break repair and homologous recombination in Escherichia coli proceed by the RecBCD pathway, which is regulated by cis-acting elements known as chi sites. A crucial feature of this regulation is the RecBCD enzyme-directed loading of RecA protein specifically onto the 3'-terminal, chi-containing DNA strand. Here we show that RecBC enzyme (lacking the RecD subunit) loads RecA protein constitutively onto the 3'-terminal DNA strand, with no requirement for chi. This strand is preferentially utilized in homologous pairing reactions. We propose that RecA protein loading is a latent property of the RecBCD holoenzyme, which is normally blocked by the RecD subunit and is revealed following interaction with chi.

Blotting, Southern↗
PubMed1991

Linkage distortion following conjugational transfer of sbcC+ to recBC sbcBC strains of Escherichia coli.

Conjugational recombination in Escherichia coli depends normally on RecBCD enzyme, a multifunctional nuclease and DNA helicase produced by the recB, recC, and recD genes. However, recombination can proceed efficiently without RecBCD in recB or recC strains carrying additional mutations in both the sbcB and sbcC genes. Recombination in these strains, sometimes referred to as the RecF pathway, requires gene products that are not essential in the RecBCD-dependent process predominating in the wild type. It has also been reported to produce a different spectrum of recombinant genotypes in crosses with Hfr donors. However, the sbcC+ gene was unknowingly transferred to the recipient strain in some of these crosses, and this may have affected the outcome. This possibility was examined by conducting parallel crosses with Hfr donors that were either wild type or mutant for sbcC. Transfer of sbcC+ from an Hfr donor is shown to alter the frequency of recombinant genotypes recovered. There is a severe reduction in progeny that inherit donor markers linked to the sbcC+ allele and an increase in the incidence of multiple exchanges. Colonies of mixed genotype for one or more of the unselected proximal markers are also much more prevalent. Since the yield of recombinants is lower than normal, these changes are attributed to the reduced viability of recombinants that inherit sbcC+ from the Hfr donor. When the Hfr donor used is also mutant for sbcC, the yield of recombinants is greater and the frequencies of the different genotypes recovered are similar to those obtained in crosses with a rec+ sbc+ recipient, in which transfer of sbcC+ has no apparent effect. Earlier studies are re-examined in light of these findings. It is concluded that, while recombination in recBC sbcBC strains involves different enzymes, the underlying molecular mechanism is essentially the same as that in the wild type.

Chromosomes, Bacterial↗
PubMed1993

Genetic analysis of double-strand break repair in Escherichia coli.

We had reported that a double-strand gap (ca. 300 bp long) in a duplex DNA is repaired through gene conversion copying a homologous duplex in a recB21 recC22 sbcA23 strain of Escherichia coli, as predicted on the basis of the double-strand break repair models. We have now examined various mutants for this repair capacity. (i) The recE159 mutation abolishes the reaction in the recB21C22 sbcA23 background. This result is consistent with the hypothesis that exonuclease VIII exposes a 3'-ended single strand from a double-strand break. (ii) Two recA alleles, including a complete deletion, fail to block the repair in this recBC sbcA background. (iii) Mutations in two more SOS-inducible genes, recN and recQ, do not decrease the repair. In addition, a lexA (Ind-) mutation, which blocks SOS induction, does not block the reaction. (iv) The recJ, recF, recO, and recR gene functions are nonessential in this background. (v) The RecBCD enzyme does not abolish the gap repair. We then examined genetic backgrounds other than recBC sbcA, in which the RecE pathway is not active. We failed to detect the double-strand gap repair in a rec+, a recA1, or a recB21 C22 strain, nor did we find the gap repair activity in a recD mutant or in a recB21 C22 sbcB15 sbcC201 mutant. We also failed to detect conservative repair of a simple double-strand break, which was made by restriction cleavage of an inserted linker oligonucleotide, in these backgrounds. We conclude that the RecBCD, RecBCD-, and RecF pathways cannot promote conservative double-strand break repair as the RecE and lambda Red pathways can.

Bacterial Proteins↗
PubMed1996

The C terminus of the AddA subunit of the Bacillus subtilis ATP-dependent DNase is required for the ATP-dependent exonuclease activity but not for the helicase activity.

Comparison of subunit AddA of the Bacillus subtilis AddAB enzyme, subunit RecB of the Escherichia coli RecBCD enzyme, and subunit RecB of the Haemophilus influenzae RecBCD enzyme revealed several regions of homology. Whereas the first seven regions are common among helicases, the two C-terminally located regions are unique for RecB of E. coli and H. influenzae and AddA. Deletion of the C-terminal region resulted in the production of an enzyme which showed moderately impaired levels of ATP-dependent helicase activity, whereas the ATP-dependent exonuclease activity was completely destroyed. The mutant enzyme was almost completely capable of complementing E. coli recBCD and B. subtilis addAB strains with respect to DNA repair and homologous recombination. These results strongly suggest that at least part of the C-terminal region of the AddA protein is indispensable for exonuclease activity and that, in contrast to the exonuclease activity, the helicase activity of the addAB gene product is important for DNA repair and homologous recombination.

Amino Acid Sequence↗
PubMed2005

A defect in the acetyl coenzyme A<-->acetate pathway poisons recombinational repair-deficient mutants of Escherichia coli.

Recombinational repair-dependent mutants identify ways to avoid chromosomal lesions. Starting with a recBC(Ts) strain of Escherichia coli, we looked for mutants unable to grow at 42 degrees C in conditions that inactivate the RecBCD(Ts) enzyme. We isolated insertions in ackA and pta, which comprise a two-gene operon responsible for the acetate<-->acetyl coenzyme A interconversion. Using precise deletions of either ackA or pta, we showed that either mutation makes E. coli cells dependent on RecA or RecBCD enzymes at high temperature, suggesting dependence on recombinational repair rather than on the RecBCD-catalyzed linear DNA degradation. Complete inhibition of growth of pta/ackA rec mutants was observed only in the presence of nearby growing cells, indicating cross-inhibition. pta rec mutants were sensitive to products of the mixed-acid fermentation of pyruvate, yet none of these substances inhibited growth of the double mutants in low-millimolar concentrations. pta, but not ackA, mutants also depend on late recombinational repair functions RuvABC or RecG. pta/ackA recF mutants are viable, suggesting, together with the inviability of pta/ackA recBC mutants, that chromosomal lesions due to the pta/ackA defect are of the double-strand-break type. We have isolated three insertional suppressors that allow slow growth of pta recBC(Ts) cells under nonpermissive conditions; all three are in or near genes with unknown functions. Although they do not form colonies, ackA rec and pta rec mutants are not killed under the nonpermissive conditions, exemplifying a case of synthetic inhibition rather than synthetic lethality.

Acetic Acid↗
PubMed2000

The Bacillus subtilis AddAB helicase/nuclease is regulated by its cognate Chi sequence in vitro.

The AddAB enzyme is important to homologous DNA recombination in Bacillus subtilis, where it is thought to be the functional counterpart of the RecBCD enzyme of Escherichia coli. In vivo, AddAB responds to a specific five-nucleotide sequence (5'-AGCGG-3' or its complement) in a manner analogous to the response of the RecBCD enzyme to interaction with chi sequences. Here, we show that purified AddAB enzyme is able to load at a double-stranded DNA end and is both a DNA helicase and nuclease, whose combined action results in the degradation of both strands of the DNA duplex. During translocation, recognition of the properly oriented sequence 5'-AGCGG-3' causes attenuation of the AddAB enzyme nuclease activity that is responsible for degradation of the strand 3'-terminal at the entry site. Therefore, we conclude that 5'-AGCGG-3' is the B. subtilis Chi site and it is hereafter referred to as chi(Bs). After encountering chi(Bs), both the degradation of the 5'-terminal strand and the helicase activity persist. Thus, processing of a double-stranded DNA end by the AddAB enzyme produces a duplex DNA molecule with a protruding 3'-terminated single-stranded tail, a universal intermediate of the recombination process.

Attachment Sites, Microbiological↗