recBC Mutants Research Articles

Essential Role for an Isoform of Escherichia coli Translation Initiation Factor IF2 in Repair of Two-Ended DNA Double-Strand Breaks.

In Escherichia coli, three isoforms of the essential translation initiation factor IF2 (IF2-1, IF2-2, and IF2-3) are generated from separate in-frame initiation codons in infB. The isoforms have earlier been suggested to additionally participate in DNA damage repair and replication restart. It is also known that the proteins RecA and RecBCD are needed for repair of DNA double-strand breaks (DSBs) in E. coli. Here, we show that strains lacking IF2-1 are profoundly sensitive to two-ended DSBs in DNA generated by radiomimetic agents phleomycin or bleomycin, or by endonuclease I-SceI. However, these strains remained tolerant to other DSB-generating genotoxic agents or perturbations to which recA and recBC mutants remained sensitive, such as to mitomycin C, type-2 DNA topoisomerase inhibitors, or DSB caused by palindrome cleavage behind a replication fork. Data from genome-wide copy number analyses following I-SceI cleavage at a single chromosomal locus suggested that, in a strain lacking IF2-1, the magnitude of recombination-dependent replication through replication restart mechanisms is largely preserved but the extent of DNA resection around the DSB site is reduced. We propose that in the absence of IF2-1 it is the synapsis of a RecA nucleoprotein filament to its homologous target that is weakened, which in turn leads to a specific failure in assembly of Ter-to-oriC directed replisomes needed for consummation of two-ended DSB repair. IMPORTANCE Double-strand breaks (DSBs) in DNA are major threats to genome integrity. In Escherichia coli, DSBs are repaired by RecA- and RecBCD-mediated homologous recombination (HR). This study demonstrates a critical role for an isoform (IF2-1) of the translation initiation factor IF2 in the repair of two-ended DSBs in E. coli (that can be generated by ionizing radiation, certain DNA-damaging chemicals, or endonuclease action). It is proposed that IF2-1 acts to facilitate the function of RecA in the synapsis between a pair of DNA molecules during HR.

Broken replication forks trigger heritable DNA breaks in the terminus of a circular chromosome.

It was recently reported that the recBC mutants of Escherichia coli, deficient for DNA double-strand break (DSB) repair, have a decreased copy number of their terminus region. We previously showed that this deficit resulted from DNA loss after post-replicative breakage of one of the two sister-chromosome termini at cell division. A viable cell and a dead cell devoid of terminus region were thus produced and, intriguingly, the reaction was transmitted to the following generations. Using genome marker frequency profiling and observation by microscopy of specific DNA loci within the terminus, we reveal here the origin of this phenomenon. We observed that terminus DNA loss was reduced in a recA mutant by the double-strand DNA degradation activity of RecBCD. The terminus-less cell produced at the first cell division was less prone to divide than the one produced at the next generation. DNA loss was not heritable if the chromosome was linearized in the terminus and occurred at chromosome termini that were unable to segregate after replication. We propose that in a recB mutant replication fork breakage results in the persistence of a linear DNA tail attached to a circular chromosome. Segregation of the linear and circular parts of this “σ-replicating chromosome” causes terminus DNA breakage during cell division. One daughter cell inherits a truncated linear chromosome and is not viable. The other inherits a circular chromosome attached to a linear tail ending in the chromosome terminus. Replication extends this tail, while degradation of its extremity results in terminus DNA loss. Repeated generation and segregation of new σ-replicating chromosomes explains the heritability of post-replicative breakage. Our results allow us to determine that in E. coli at each generation, 18% of cells are subject to replication fork breakage at dispersed, potentially random, chromosomal locations.

Replication Forks Stalled at Ultraviolet Lesions Are Rescued via RecA and RuvABC Protein-catalyzed Disintegration in Escherichia coli

Ultraviolet (UV) irradiation is not known to induce chromosomal fragmentation in sublethal doses, and yet UV irradiation causes genetic instability and cancer, suggesting that chromosomes are fragmented. Here we show that UV irradiation induces fragmentation in sublethal doses, but the broken chromosomes are repaired or degraded by RecBCD; therefore, to observe full fragmentation, RecBCD enzyme needs to be inactivated. Using quantitative pulsed field gel electrophoresis and sensitive DNA synthesis measurements, we investigated the mechanisms of UV radiation-induced chromosomal fragmentation in recBC mutants, comparing five existing models of DNA damage-induced fragmentation. We found that fragmentation depends on active DNA synthesis before, but not after, UV irradiation. At low UV irradiation doses, fragmentation does not need excision repair or daughter strand gap repair. Fragmentation absolutely depends on both RecA-catalyzed homologous strand exchange and RuvABC-catalyzed Holliday junction resolution. Thus, chromosomes fragment when replication forks stall at UV lesions and regress, generating Holliday junctions. Remarkably, cells specifically utilize fork breakage to rescue stalled replication and avoid lethality.

RecA433 cells are defective in recF-mediated processing of disrupted replication forks but retain recBCD-mediated functions

RecA is required for recombinational processes and cell survival following UV-induced DNA damage. recA433 is a historically important mutant allele that contains a single amino acid substitution (R243H). This mutation separates the recombination and survival functions of RecA. recA433 mutants remain proficient in recombination as measured by conjugation or transduction, but are hypersensitive to UV-induced DNA damage. The cellular functions carried out by RecA require either recF pathway proteins or recBC pathway proteins to initiate RecA-loading onto the appropriate DNA substrates. In this study, we characterized the ability of recA433 to carry out functions associated with either the recF pathway or recBC pathway. We show that several phenotypic deficiencies exhibited by recA433 mutants are similar to recF mutants but distinct from recBC mutants. In contrast to recBC mutants, recA433 and recF mutants fail to process or resume replication following disruption by UV-induced DNA damage. However, recA433 and recF mutants remain proficient in conjugational recombination and are resistant to formaldehyde-induced protein–DNA crosslinks, functions that are impaired in recBC mutants. The results are consistent with a model in which the recA433 mutation selectively impairs RecA functions associated with the RecF pathway, while retaining the ability to carry out RecBCD pathway-mediated functions. These results are discussed in the context of the recF and recBC pathways and the potential substrates utilized in each case.

RdgB acts to avoid chromosome fragmentation in Escherichia coli.

Bacterial RecA protein is required for repair of two-strand DNA lesions that disable whole chromosomes. recA mutants are viable, suggesting a considerable cellular capacity to avoid these chromosome-disabling lesions. recA-dependent mutants reveal chromosomal lesion avoidance pathways. Here we characterize one such mutant, rdgB/yggV, deficient in a putative inosine/xanthosine triphosphatase, conserved throughout kingdoms of life. The rdgB recA lethality is suppressed by inactivation of endonuclease V (gpnfi) specific for DNA-hypoxanthines/xanthines, suggesting that RdgB either intercepts improper DNA precursors dITP/dXTP or works downstream of EndoV in excision repair of incorporated hypoxathines/xanthines. We find that DNA isolated from rdgB mutants contains EndoV-recognizable modifications, whereas DNA from nfi mutants does not, substantiating the dITP/dXTP interception by RdgB. rdgB recBC cells are inviable, whereas rdgB recF cells are healthy, suggesting that chromosomes in rdgB mutants suffer double-strand breaks. Chromosomal fragmentation is indeed observed in rdgB recBC mutants and is suppressed in rdgB recBC nfi mutants. Thus, one way to avoid chromosomal lesions is to prevent hypoxanthine/xanthine incorporation into DNA via interception of dITP/dXTP.

Chromosomal lesion suppression and removal in Escherichia coli via linear DNA degradation.

RecBCD is a DNA helicase/exonuclease implicated in degradation of foreign linear DNA and in RecA-dependent recombinational repair of chromosomal lesions in E. coli. The low viability of recA recBC mutants vs. recA mutants indicates the existence of RecA-independent roles for RecBCD. To distinguish among possible RecA-independent roles of the RecBCD enzyme in replication, repair, and DNA degradation, we introduced wild-type and mutant combinations of the recBCD chromosomal region on a low-copy-number plasmid into a DeltarecA DeltarecBCD mutant and determined the viability of resulting strains. Our results argue against ideas that RecBCD is a structural element in the replication factory or is involved in RecA-independent repair of chromosomal lesions. We found that RecBCD-catalyzed DNA degradation is the only activity important for the recA-independent viability, suggesting that degradation of linear tails of sigma-replicating chromosomes could be one of the RecBCD's roles. However, since the weaker DNA degradation capacity due a combination of the RecBC helicase and ssDNA-specific exonucleases restores viability of the DeltarecA DeltarecBCD mutant to a significant extent, we favor suppression of chromosomal lesions via linear DNA degradation at reversed replication forks as the major RecA-independent role of the RecBCD enzyme.

Role of the RecBCD recombination pathway in Salmonella virulence.

Mutants of Salmonella enterica lacking the RecBC function are avirulent in mice and unable to grow inside macrophages (N. A. Buchmeier, C. J. Lipps, M. Y. H. So, and F. Heffron, Mol. Microbiol. 7:933-936, 1993). The virulence-related defects of RecBC(-) mutants are not suppressed by sbcB and sbcCD mutations, indicating that activation of the RecF recombination pathway cannot replace the virulence-related function(s) of RecBCD. Functions of the RecF pathway such as RecJ and RecF are not required for virulence. Since the RecBCD pathway, but not the RecF pathway, is known to participate in the repair of double-strand breaks produced during DNA replication, we propose that systemic infection by S. enterica may require RecBCD-mediated recombinational repair to prime DNA replication inside phagocytes. Mutants lacking both RecD and RecJ are also attenuated in mice and are unable to proliferate in macrophages, suggesting that exonucleases V and IX provide alternative functions for RecBCD-mediated recombinational repair during Salmonella infection.

SbcB sbcC null mutations allow RecF-mediated repair of arrested replication forks in rep recBC mutants.

We have proposed previously that, in Escherichia coli, blockage of replication forks can lead to the reversal of the fork. Annealing of the newly synthesized strands creates a double-stranded end adjacent to a Holliday junction. The junction is migrated away from the DNA end by RuvAB and can be cleaved by RuvC, while RecBCD is required for the repair of the double-stranded tail. Consequently, the rep mutant, in which replication arrests are frequent and fork reversal occurs, requires RecBCD for growth. We show here that the combination of sbcB sbcCD null mutations restores the viability to rep recBC mutants by activation of the RecF pathway of recombination. This shows that the proteins belonging to the RecF pathway are able to process the DNA ends made by the replication fork reversal into a structure that allows recombination-dependent replication restart. However, we confirm that, unlike sbcB null mutations, sbcB15, which suppresses all other recBC mutant defects, does not restore the viability of rep recBC sbcCD strains. We also show that ruvAB inactivation suppresses the lethality and the formation of double-stranded breaks (DSBs) in a rep recBC recF strain, totally deficient for homologous recombination, as well as in rep recBC mutants. This confirms that RuvAB processing of arrested replication forks is independent of the presence of recombination intermediates.

DNA double-strand breaks caused by replication arrest.

We report here that DNA double-strand breaks (DSBs) form in Escherichia coli upon arrest of replication forks due to a defect in, or the inhibition of, replicative DNA helicases. The formation of DSBs was assessed by the appearance of linear DNA detected by pulse-field gel electrophoresis. Processing of DSBs by recombination repair or linear DNA degradation was abolished by mutations in recBCD genes. Two E. coli replicative helicases were tested, Rep, which is essential in recBC mutants, and DnaB. The proportion of linear DNA increased up to 50% upon shift of rep recBTS recCTS cells to restrictive temperature. No increase in linear DNA was observed in the absence of replicating chromosomes, indicating that the formation of DSBs in rep strains requires replication. Inhibition of the DnaB helicase either by a strong replication terminator or by a dnaBTS mutation led to the formation of linear DNA, showing that blocked replication forks are prone to DSB formation. In wild-type E. coli, linear DNA was detected in the absence of RecBC or of both RecA and RecD. This reveals the existence of a significant amount of spontaneous DSBs. We propose that some of them may also result from the impairment of replication fork progression.

Lethality of rep recB and rep recC double mutants of Escherichia coli.

A rep mutation in combination with a recB or a recC mutation renders Escherichia coli non-viable. This conclusion is based on the following lines of evidence: (i) double mutants cannot be constructed by P1 transduction; (ii) induction of the lambda Gam protein, which inactivates most of the RecBCD activities, is lethal in rep mutants; (iii) rep recBts recCts mutants are not viable at high temperature. The resons for a requirement for the RecBCD enzyme in rep strains were investigated. Initiation of chromosome replication, elongation and chromosomal segregation do not seem impaired in the rep recBtsrecCts mutant at the non-permissive temperature. The viability of other rep derivatives was tested. rep recA recD triple mutants are not viable, whereas rep recD and rep recA double mutants are. Inactivation of both exoV activity and recBC-dependent homologous recombination is therefore responsible for the non-viability of rep recBC strains. However, sbcA and sbcB mutations, which render recBC mutants recombination proficient, do not restore viability of rep recBC mutants, indicating that recombination via the RecF or the RecE pathways cannot functionally replace RecBCD-mediated recombination. The specific requirement for RecBCD suggests the occurrence of double-strand DNA breaks in rep strains. Additional arguments in favour of the presence of DNA lesions in rep mutants are as follows: (i) expression of SOS repair functions delays lethality of rep derivatives after inactivation of RecBCD; (ii) sensitivity of rep strains to ultraviolet light is increased by partial inactivation of RecBCD. A model for the recovery of cells from double-strand breaks in rep mutants is discussed.

Homologous recombination-dependent initiation of DNA replication from DNA damage-inducible origins in Escherichia coli.

Escherichia coli cells induced for the SOS response express inducible stable DNA replication (iSDR) as an SOS function. Initiation of iSDR is independent of transcription, translation and DnaA protein, which are essential for initiation of DNA replication from oriC. We found that a recA mutant that is defective in recombination but proficient in SOS induction could not elicit iSDR. In contrast, iSDR was enhanced by recD and recJ mutations that inactivate the exonuclease V activity of the RecBCD enzyme and the RecJ exonuclease activity, respectively. A mutation in the ruvC gene that blocks the resolution of recombination intermediates (i.e. Holliday structures) also enhanced iSDR. Furthermore, inhibition of branch migration by recG or ruvAB mutations dramatically increased the iSDR activity. recBC mutants are defective in iSDR induction but the defect was suppressed by a mutation in the sbcA gene. The major product of minichromosomes replicated by iSDR was covalently closed circular monomers. We propose that recombination intermediates (i.e. D-loop structures) created by the action of RecA recombinase and RecBC(D) helicase play a central role in initiation of iSDR.

Properties of Escherichia coli expressing bacteriophage P22 Abc (anti-RecBCD) proteins, including inhibition of Chi activity

Escherichia coli strains bearing plasmids expressing phage P22 anti-RecBCD functions abc1 and abc2 were tested for the presence of recBC-like phenotypes. Abc2 induces moderate sensitivity to UV light in wild-type and recD mutant strains but severely sensitizes both recF and recJ mutants. Abc1 has little effect on UV sensitivity in wild-type or recF or recJ mutant hosts but increases the sensitivity of recD mutants to a UV dose of 20 J/m2 about 10-fold. Abc2 induces E. coli to segregate inviable cells during growth, interferes with the growth of lambda red gam chi+ and chi 0 phage (the effect is greater with chi+ phage), inhibits Chi and Chi-like activity as measured by lambda red gam crosses, and prevents SOS induction in response to nalidixic acid; Abc1 has no effect in these tests. Abc2, alone or with Abc1, does not allow the growth of lambda red gam in the presence of a P2 prophage but does not kill the P2 lysogenic host (as lambda Gam does). Finally, Abc2 inhibits conjugational recombination in wild-type cells to the level seen in recBC mutants. These data suggest that Abc2 inhibits the recombination-promoting ability of RecBCD but leaves the exonuclease functions intact.

Escherichia coli xonA (sbcB) mutants enhance illegitimate recombination.

Mutations of Escherichia coli K-12 were isolated that increase the frequency of deletion formation. Three of these mutations map to the gene sbcB at 43.5 min on the E. coli chromosome. Two types of mutations at sbcB have been previously defined: sbcB-type that suppress both the UV sensitivity and recombination deficiency of recBC mutants, and xonA-type that suppress only the UV sensitivity. Both types are defective for production of exonuclease I activity. The mutations isolated here were similar to xonA alleles of sbcB because they suppressed the UV sensitivity of recBC mutants but did not restore recombination proficiency. Indeed, two previously characterized xonA alleles were shown to increase the frequency of deletion formation, although an sbcB allele did not. This result demonstrates that loss of exonuclease I activity is not sufficient to confer a high deletion phenotype, rather, the product of the sbcB gene possesses some other function that is important for deletion formation. Because deletion formation in this system is recA independent and does not require extensive DNA homology, these mutations affect a pathway of illegitimate recombination.

DNA repair mechanisms affecting cytotoxicity by streptozotocin in E. coli

Mechanisms underlying cytotoxicity by the monofunctional nitrosourea streptozotocin (STZ) were evaluated in DNA repair-deficient E. coli mutants. Strains not proficient in recombinational repair which lack either RecA protein or ReBC gene products were highly sensitive to STZ. In contrast, cells that constitutively synthesize RecA protein and cannot initiate SOS repair mechanisms because of uncleavable LexA repressor (recAo98 lexA3) were resistant to this drug compared to a lexA3 strain. Further, E. coli cells lacking both 3-methyladenine DNA glycosylases I (tag) and II (alkA) also were highly sensitive to STZ. DNA synthesis was most inhibited by STZ in recA and alkA tag E. coli mutants, but was suppressed less markedly in wild-type and recBC cells. DNA degradation was most extensive in recA E. coli after STZ treatment, while comparable in recBC, alkA tag, and wild-type cells. Although increased single-stranded DNA breals were present after STZ treatment in recA and recBC mutants compared to the wild type, no significant increase in DNA single-stranded breaks was noted in alkA tag E. coli. Further, DNA breaks in recBC cells were repaired, while those present in recA cells were not. These findings establish the critical importance of both recombinational repair and 3-methyladenine DNA glycosylase in ameliorating cytotoxic effects and DNA damage caused by STZ in E. coli.

Inhibition of the SOS response of Escherichia coli by the Ada protein.

Induction of the adaptive response by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused a decrease in the UV-mediated expression of both recA and sfiA genes but not of the umuDC gene. On the other hand, the adaptive response did not affect the temperature-promoted induction of SOS response in a RecA441 mutant. The inhibitory effect on the UV-triggered expression of the recA and sfiA genes was not dependent on either the alkA gene or the basal level of RecA protein, but rather required the ada gene. Furthermore, an increase in the level of the Ada protein, caused by the runaway plasmid pYN3059 in which the ada gene is regulated by the lac promoter, inhibited UV-mediated recA gene expression even in cells to which the MNNG-adaptive treatment had not been applied. This inhibitory effect of the adaptive pretreatment was not observed either in RecBC- strains or in RecBC mutants lacking exonuclease V-related nuclease activity. However, RecF- mutants showed an adaptive response-mediated decrease in UV-promoted induction of the recA gene.

Further characterization of SOS system induction in recBC mutants of Escherichia coli

Expression of several SOS functions such as induction of lambda prophage, inhibition of cell division and induction of both umuC and recA genes after UV-irradiation, nalidixic acid or mitomycin C addition was studied in an RecBC − mutant. UV-irradiation and mitomycin C induced all SOS functions studied in the RecBC − cells but at a lower level and delayed with respect to the wild-type strain. On the contrary, nalidixic acid was unable to trigger any of these SOS functions. In the RecBC − mutant, adenine only had a stimulating effect on the amplification of RecA protein synthesis following UV-irradiation. Nevertheless, in the wild-type strain the stimulating effect occurred in all SOS functions studied following UV-irradiation as well as in the amplification of RecA protein synthesis by nalidixic acid but not in the other SOS functions triggered by this compound. Furthermore, adenine produced a decrease in the mitomycin C-mediated induction of all SOS functions studied in both RecBC − and wild-type strains.

A new class of Escherichia coli recBC mutants: implications for the role of RecBC enzyme in homologous recombination.

Mutants of Escherichia coli sensitive to phage T4 gene 2 mutants were obtained following ethyl methanesulfonate mutagenesis. By mapping and complementation analysis, the mutations in each of the six mutants are in recB and recC. By both in vivo and in vitro analyses, the nuclease activity of RecBC enzyme is undetectable in these mutants. However, by several other criteria, such as proficiency in recombination, relative resistance to UV radiation, and viability of the cells in the culture, these mutants are almost identical to their recBC+ parent. The properties of these mutants indicate that the ATP-dependent double-stranded DNA exonuclease activity of RecBC enzyme is not required for recombination. Chi recombinational hotspots, which stimulate recombination by the RecBC pathway, have no detectable activity in the mutants. This result suggests that the nuclease activity of RecBC enzyme is required for Chi activity and is consistent with the hypothesis that Chi stimulates recombination by directing RecBC enzyme to cut DNA at or near Chi.

Genetic analysis and regulation of inducible recombination in Escherichia coli K-12.

The highly efficient conjugational process of genetic exchange in Escherichia coli has provided a useful model system for investigating recombination between homologous DNA molecules. In standard Hfr × F− crosses, the formation of viable recombinants normally requires the products of recB and recC, which together specify a DNA dependent ATPase that functions both as a nuclease and as a DNA helicase (Telander Muskavitch and Linn 1981). An alternative, recBC-independent mechanism can be activated in recBC mutants by the additional mutation of sbcB, the structural gene for exonuclease I (Kushner et al. 1971, 1972). This depends on the function of recF, recJ, recN, and ruv products, none of which is essential in the presence of RecBC enzyme (Horii and Clark 1973; Lloyd et al. 1983, 1984; Lovett and Clark 1984).

Thymineless death in recombination deficient mutants of Escherichia coli K12.

When grown in thymine-free minimal synthetic medium, thyA recA mutants of Escherichia coli K12 were comparatively more resistant and thyA recBC mutants more sensitive to thymineless death (TLD) than their respective parent strains. No excessive numbers of single-strand breaks were observed in the DNA of the recBC mutant strain starved for thymine, hence the hypersensitivity for TLD in this strain was not caused by this type of DNA damage. Although experiments were performed with the same recBC mutant strain as used by earlier workers, results presented here contradict earlier findings that recBC mutants are no more sensitive and recA mutants are no more resistant to TLD.

Restriction in vivo. V. Introduction of SOS functions in Escherichia coli by restricted T4 phage DNA, and alleviation of restriction by SOS functions.

Degradation products of restricted T4 DNA induced filamentation, mutagenesis, and to a lesser extent, synthesis of recA protein in wild type cells but not in recA, lexA or recBC mutants of Escherichia coli. We conclude that the structural damage to the DNA caused by restriction cleavage and exonuclease V degradation can induce SOS functions. Degradation of restricted nonglycosylated T4 DNA by exonuclease V delayed cell division and induced filament formation and mutagenesis in lexA+ but not in lexA- cells. Delay of cell division was also dependent upon recA and recBC functions. Such degradation of DNA also dramatically increased mutagenesis in tif- Sfi- cells at 42 degrees. The synthesis of recA protein continued in the restricting host after infection by the monglucosylated T4 phage, but enhanced synthesis is not induced to the extent seen in SOS induced tif- cells grown at 42 degrees. We also found that restriction of nonglycosylated T4 was alleviated in UV irradiated cells. The UV induced alleviation of rgl and rK restriction depended upon post irradiation protein synthesis and was not observed in recA, lexA or recBC mutants.