RecBCD coordinates repair of two ends at a DNA double-strand break, preventing aberrant chromosome amplification.

DNA double-strand break (DSB) repair is critical for cell survival. A diverse range of organisms from bacteria to humans rely on homologous recombination for accurate DSB repair. This requires both coordinate action of the two ends of a DSB and stringent control of the resultant DNA replication to prevent unwarranted DNA amplification and aneuploidy. In Escherichia coli, RecBCD enzyme is responsible for the initial steps of homologous recombination. Previous work has revealed recD mutants to be nuclease defective but recombination proficient. Despite this proficiency, we show here that a recD null mutant is defective for the repair of a two-ended DSB and that this defect is associated with unregulated chromosome amplification and defective chromosome segregation. Our results demonstrate that RecBCD plays an important role in avoiding this amplification by coordinating the two recombining ends in a manner that prevents divergent replication forks progressing away from the DSB site.

[Role of double strand DNA break repair for quinolone sensitivity in Escherichia coli: therapeutic implications]. / El papel de la reparación de roturas de doble cadena de ADN en Escherichia coli en la sensibilidad a quinolonas: implicaciones terapéuticas.

INTRODUCTION: Quinolones are one of the types of antibiotics with higher resistance rates in the last years. At molecular level, quinolones block type II topoisomerases producing double strand breaks (DSBs). These DSBs could play a double role, as inductors of the quinolone bactericidal effects but also as mediators of the resistance and tolerance mechanisms. MATERIAL AND METHODS: In this work we have studied the molecular pathways responsible for DSBs repair in the quinolone susceptibility: the stalled replication fork reversal (recombination-dependent) (RFR), the SOS response induction, the translesional DNA synthesis (TLS) and the nucleotide excision repair mechanisms (NER). For this reason, at the European University in Madrid, we analysed the minimal inhibitory concentration (MIC) to three different quinolones in Escherichia coli mutant strains coming from different type culture collections. RESULTS: recA, recBC, priA and lexA mutants showed a significant reduction on the MIC values for all quinolones tested. No significant changes were observed on mutant strains for TLS and NER. DISCUSSION: These data indicate that in the presence of quinolones, RFR mechanisms and the SOS response could be involved in the quinolone susceptibility.

Control of RecBCD enzyme activity by DNA binding- and Chi hotspot-dependent conformational changes.

Faithful repair of DNA double-strand breaks by homologous recombination is crucial to maintain functional genomes. The major Escherichia coli pathway of DNA break repair requires RecBCD enzyme, a complex protein machine with multiple activities. Upon encountering a Chi recombination hotspot (5' GCTGGTGG 3') during DNA unwinding, RecBCD's unwinding, nuclease, and RecA-loading activities change dramatically, but the physical basis for these changes is unknown. Here, we identify, during RecBCD's DNA unwinding, two Chi-stimulated conformational changes involving RecC. One produced a marked, long-lasting, Chi-dependent increase in protease sensitivity of a small patch, near the Chi recognition domain, on the solvent-exposed RecC surface. The other change was identified by crosslinking of an artificial amino acid inserted in this RecC patch to RecB. Small-angle X-ray scattering analysis confirmed a major conformational change upon binding of DNA to the enzyme and is consistent with these two changes. We propose that, upon DNA binding, the RecB nuclease domain swings from one side of RecC to the other; when RecBCD encounters Chi, the nuclease domain returns to its initial position determined by crystallography, where it nicks DNA exiting from RecC and loads RecA onto the newly generated 3'-ended single-stranded DNA during continued unwinding; a crevice between RecB and RecC increasingly narrows during these steps. This model provides a physical basis for the intramolecular "signal transduction" from Chi to RecC to RecD to RecB inferred previously from genetic and enzymatic analyses, and it accounts for the enzymatic changes that accompany Chi's stimulation of recombination.

One motor driving two translocases.

In this issue, Wu et al. show that the RecBC helicase, which is involved in repairing double-strand DNA breaks,uses one ATPase motor to drive two translocases along opposite strands of DNA­much as an all-wheel drive engine controls movement of both front and back wheels. This mechanism may allow RecBC to load onto blunt-end DNA more efficiently and to move through obstacles such as gaps and DNA damage.

Escherichia coli RecBC helicase has two translocase activities controlled by a single ATPase motor.

E. coli RecBCD is a DNA helicase with two ATPase motors (RecB, a 3'→5' translocase, and RecD, a 5'→3' translocase) that function in repair of double-stranded DNA breaks. The RecBC heterodimer, with only the RecB motor, remains a processive helicase. Here we examined RecBC translocation along single-stranded DNA (ssDNA). Notably, we found RecBC to have two translocase activities: the primary translocase moves 3'→5', whereas the secondary translocase moves RecBC along the opposite strand of a forked DNA at a similar rate. The secondary translocase is insensitive to the ssDNA backbone polarity, and we propose that it may fuel RecBCD translocation along double-stranded DNA ahead of the unwinding fork and ensure that the unwound single strands move through RecBCD at the same rate after interaction with a crossover hot-spot indicator (Chi) sequence.

Phylogenetic ubiquity and shuffling of the bacterial RecBCD and AddAB recombination complexes.

RecBCD and AddAB are bacterial enzymes that share similar helicase and nuclease activities and initiate repair of DNA double-strand breaks by homologous recombination. Examination of the phylogenetic distribution of AddAB and RecBCD revealed that one or the other complex is present in most sequenced bacteria. In addition, horizontal gene transfer (HGT) events involving addAB and recBCD appear to be common, with the genes encoding one complex frequently replacing those encoding the other. HGT may also explain the unexpected identification of archaeal addAB genes. More than 85% of addAB and recBCD genes are clustered on the genome, suggesting operon structures. A few organisms, including the Mycobacteria, encode multiple copies of these complexes of either the same or mixed classes. The possibility that the enzymatic activities of the AddAB and RecBCD enzymes promote their horizontal transfer is discussed, and the distribution of AddAB/RecBCD is compared to that of the RecU/RuvC resolvases. Finally, it appears that two sequence motifs, the Walker A box involved in ATP binding and an iron-sulfur-cysteine cluster, are present only in subsets of AddB proteins, suggesting the existence of mechanistically distinct classes of AddB.

Construction of recB-recD genetic fusion and functional analysis of RecBDC fusion enzyme in Escherichia coli.

BACKGROUND: recD, located between recB and argA, encodes the smallest polypeptide (60 kDa) of the heterotrimeric enzyme RecBCD in Escherichia coli. RecD is a 5'-3' helicase and is required for the nuclease activity of RecBCD and for tight binding to dsDNA ends. Here, we have tested the hypothesis that RecD regulates the structure and activities of RecBCD, including RecA loading. RESULTS: To characterize its regulatory functions, recD was genetically fused to recB through deletion and substitution mutations. The recB-recD fusion led to a decreased amount of the heterotrimer. Both fusion mutants proved to be recombination proficient, viable and resistant to DNA damaging agents, and to have DNA unwinding, ATP-dependent dsDNA exonuclease and Chi genetic activities. CONCLUSION: Our findings suggest that the recB-recD fusion may form a RecBD fusion protein and therefore affect RecD assembly, but this does not change the three-dimensional structure of the heterotrimer.

In vivo role of Escherichia coli single-strand exonucleases in SOS induction by gamma radiation.

Ionizing radiation causes different types of genetic damage, ranging from base modifications to single- and double-stranded DNA breaks, which may be deleterious or even lethal to the cell. There are different repair or tolerance mechanisms to counteract the damage. Among them is the Escherichia coli SOS system: a set of genes that becomes activated upon DNA damage to confer better opportunities for cell survival. However, since this response is triggered by single-stranded DNA regions, most lesions have to be processed or modified prior to SOS activation. Several genes such as recO, recB and recJ that seem to be required to induce the response have already been reported. The results of this work indicate that the four known E.coli single-strand exonucleases take part in processing gamma radiation damage, though RecJ and ExoI proved to be more important than ExoVII or ExoX. In addition, ExoV as well as glycosylases such as Nth and, to a lesser extent, Fpg are also required. A model intended to explain the role of all these genes in damage processing is presented.

RecBCD and RecJ/RecQ initiate DNA degradation on distinct substrates in UV-irradiated Escherichia coli.

After UV irradiation, recA mutants fail to recover replication, and a dramatic and nearly complete degradation of the genomic DNA occurs. Although the RecBCD helicase/nuclease complex is known to mediate this catastrophic DNA degradation, it is not known how or where this degradation is initiated. Previous studies have speculated that RecBCD targets and initiates degradation from the nascent DNA at replication forks arrested by DNA damage. To test this question, we examined which enzymes were responsible for the degradation of genomic DNA and the nascent DNA in UV-irradiated recA cells. We show here that, although RecBCD degrades the genomic DNA after UV irradiation, it does not target the nascent DNA at arrested replication forks. Instead, we observed that the nascent DNA at arrested replication forks in recA cultures is degraded by RecJ/RecQ, similar to what occurs in wild-type cultures. These findings indicate that the genomic DNA degradation and nascent DNA degradation in UV-irradiated recA mutants are mediated separately through RecBCD and RecJ/RecQ, respectively. In addition, they demonstrate that RecBCD initiates degradation at a site(s) other than the arrested replication fork directly.

Deletions of recBCD or recD influence genetic transformation differently and are lethal together with a recJ deletion in Acinetobacter baylyi.

In prokaryotes, homologous recombination is essential for the repair of genomic DNA damage and for the integration of DNA taken up during horizontal gene transfer. In Escherichia coli, the exonucleases RecJ (specific for 5' single-stranded DNA) and RecBCD (degrades duplex DNA) play important roles in recombination and recombinational double-strand break (DSB) repair by the RecF and RecBCD pathways, respectively. The cloned recJ of Acinetobacter baylyi partially complemented an E. coli recJ mutant, suggesting functional similarity of the enzymes. A DeltarecJ mutant of A. baylyi was only slightly altered in transformability and was not affected in UV survival. In contrast, a DeltarecBCD mutant was UV-sensitive, and had a low viability and altered transformation. Compared to wild-type, transformation with large chromosomal DNA fragments was decreased about 5-fold, while transformation with 1.5 kbp DNA fragments was increased 3.3- to 7-fold. A DeltarecD mutation did not affect transformation, viability or UV resistance. However, double mutants recJ recBCD and recJ recD were non-viable, suggesting that the RecJ DNase or the RecBCD DNase (presumably absent in recD) becomes essential for the recombinational repair of spontaneously inactivated replication forks if the other DNase is absent. A model of recombination during genetic transformation is discussed in which the two ends of the single-stranded donor DNA present in the cytoplasm frequently integrate separately and often with a time difference. If replication runs through that genomic region before both ends of the donor DNA are ligated to recipient DNA, a double-strand break (DSB) is formed. In these cases, transformation becomes dependent on DSB repair.

A new role of Deinococcus radiodurans RecD in antioxidant pathway.

In Deinococcus radiodurans, RecBCD holoenzyme is not intact because of the absence of RecB and RecC, but a RecD-like protein does indeed exist. In this work, D. radiodurans recD disruptant was constructed and its possible biological functions were investigated. The results showed that disruption of the recD gene of D. radiodurans resulted in a remarkably increased sensitivity to hydrogen peroxide but had no apparent effect on the resistance to gamma and UV radiation. Furthermore, complementation experiments showed that Escherichia coli RecD, helicase domain or N-terminal domain of D. radiodurans RecD could not individually restore the resistant phenotype to hydrogen peroxide of the recD disruptant, whereas the complete D. radiodurans RecD protein could. Further studies showed that D. radiodurans RecD took part in antioxidant process by stimulating catalase activity and reactive oxygen species scavenging activity in D. radiodurans. These results suggest that D. radiodurans RecD has a new role in the antioxidant pathway.

Neisseria gonorrhoeae DNA recombination and repair enzymes protect against oxidative damage caused by hydrogen peroxide.

The strict human pathogen Neisseria gonorrhoeae is exposed to oxidative damage during infection. N. gonorrhoeae has many defenses that have been demonstrated to counteract oxidative damage. However, recN is the only DNA repair and recombination gene upregulated in response to hydrogen peroxide (H(2)O(2)) by microarray analysis and subsequently shown to be important for oxidative damage protection. We therefore tested the importance of RecA and DNA recombination and repair enzymes in conferring resistance to H(2)O(2) damage. recA mutants, as well as RecBCD (recB, recC, and recD) and RecF-like pathway mutants (recJ, recO, and recQ), all showed decreased resistance to H(2)O(2). Holliday junction processing mutants (ruvA, ruvC, and recG) showed decreased resistance to H(2)O(2) resistance as well. Finally, we show that RecA protein levels did not increase as a result of H(2)O(2) treatment. We propose that RecA, recombinational DNA repair, and branch migration are all important for H(2)O(2) resistance in N. gonorrhoeae but that constitutive levels of these enzymes are sufficient for providing protection against oxidative damage by H(2)O(2).

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.

Effects of recJ, recQ, and recFOR mutations on recombination in nuclease-deficient recB recD double mutants of Escherichia coli.

The two main recombination pathways in Escherichia coli (RecBCD and RecF) have different recombination machineries that act independently in the initiation of recombination. Three essential enzymatic activities are required for early recombinational processing of double-stranded DNA ends and breaks: a helicase, a 5'-->3' exonuclease, and loading of RecA protein onto single-stranded DNA tails. The RecBCD enzyme performs all of these activities, whereas the recombination machinery of the RecF pathway consists of RecQ (helicase), RecJ (5'-->3' exonuclease), and RecFOR (RecA-single-stranded DNA filament formation). The recombination pathway operating in recB (nuclease-deficient) mutants is a hybrid because it includes elements of both the RecBCD and RecF recombination machineries. In this study, genetic analysis of recombination in a recB (nuclease-deficient) recD double mutant was performed. We show that conjugational recombination and DNA repair after UV and gamma irradiation in this mutant are highly dependent on recJ, partially dependent on recFOR, and independent of recQ. These results suggest that the recombination pathway operating in a nuclease-deficient recB recD double mutant is also a hybrid. We propose that the helicase and RecA loading activities belong to the RecBCD recombination machinery, while the RecJ-mediated 5'-->3' exonuclease is an element of the RecF recombination machinery.

Recombination mechanisms; fortieth anniversary meeting of the Holliday model.

The year 2004 marks the fortieth anniversary of the Holliday junction. This extraordinary DNA structure, originally proposed by Robin Holliday to explain genetic recombination in fungi, now appears to be a pivotal intermediate in many aspects of DNA metabolism. In those forty years the Holliday junction has gone from a hypothetical structure to models for its atomic structure and visualization of its dynamics at the single molecule level.