Biochemical and physical characterization of exonuclease V from Escherichia coli. Comparison of the catalytic activities of the RecBC and RecBCD enzymes

Biochemical evidence is presented that confirms exonuclease V of Escherichia coli consists of three distinct subunits encoded by the recB, recC, and recD genes. The recD gene encodes a Mr 60,000 polypeptide and physically maps 3' to the recB structural gene. The role of the recD subunit in exonuclease V function has been examined by comparing the catalytic activities of the purified RecBCD enzyme with the RecBC enzyme. The RecBC enzyme retains significant levels of DNA-dependent ATPase activity and DNA helicase activity. Endonucleolytic activity on single-stranded covalently closed DNA becomes ATP-dependent. Exonucleolytic activity on either single- and double-stranded DNA was not detected. Taken together with the phenotypic properties of recD null mutants, it appears that the exonucleolytic activities of the RecBCD enzyme are not required for genetic recombination and the repair of either UV-induced photoproducts or mitomycin C-generated DNA cross-links, but are essential for the repair of methyl methanesulfonate-induced methylation.     

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.     

Nuclease activity is essential for RecBCD recombination in Escherichia coli

RecBCD has two conflicting roles in Escherichia coli. (i) As ExoV, it is a potent double-stranded (ds)DNA exonuclease that destroys linear DNA produced by restriction of foreign DNA. (ii) As a recombinase, it promotes repair of dsDNA breaks and genetic recombination in the vicinity of chi recombination hot-spots. These paradoxical roles are accommodated by chi-dependent attenuation of RecBCD exonuclease activity and concomitant conversion of the enzyme to a recombinase. To challenge the proposal that chi converts RecBCD from a destructive exonuclease to a recombinogenic helicase, we mutated the nuclease catalytic centre of RecB and tested the resulting mutants for genetic recombination and DNA repair in vivo. We predicted that, if nuclease activity inhibits recombination and helicase activity is sufficient for recombination, the mutants would be constitutive recombinases, as has been seen in recD null mutants. Conversely, if nuclease activity is required, the mutants would be recombination deficient. Our results indicate that 5' --> 3' exonuclease activity is essential for recombination by RecBCD at chi recombination hot-spots and at dsDNA ends in recD mutants. In the absence of RecB-dependent nuclease function, recombination becomes entirely dependent on the 5' --> 3' single-stranded (ss)DNA exonuclease activity of RecJ and the helicase activity of RecBC(D).     

recD sbcB sbcD Mutants Are Deficient in Recombinational Repair of UV Lesions by RecBC

In recD sbcB sbcD mutants, repair of UV-irradiated DNA is strongly RecF dependent, indicating that RecBC is inactive. This finding suggests that exonuclease V, exonuclease I (SbcB), and the SbcCD nuclease play a redundant role in vivo, which is essential for the recombination activity of the RecBC complex during UV repair.     

DNA end resection controls the balance between homologous and illegitimate recombination in Escherichia coli

Even a partial loss of function of human RecQ helicase analogs causes adverse effects such as a cancer-prone Werner, Bloom or Rothmund-Thompson syndrome, whereas a complete RecQ deficiency in Escherichia coli is not deleterious for a cell. We show that this puzzling difference is due to different mechanisms of DNA double strand break (DSB) resection in E. coli and humans. Coupled helicase and RecA loading activities of RecBCD enzyme, which is found exclusively in bacteria, are shown to be responsible for channeling recombinogenic 3' ending tails toward productive, homologous and away from nonproductive, aberrant recombination events. On the other hand, in recB1080/recB1067 mutants, lacking RecBCD's RecA loading activity while preserving its helicase activity, DSB resection is mechanistically more alike that in eukaryotes (by its uncoupling from a recombinase polymerization step), and remarkably, the role of RecQ also becomes akin of its eukaryotic counterparts in a way of promoting homologous and suppressing illegitimate recombination. The sickly phenotype of recB1080 recQ mutant was further exacerbated by inactivation of an exonuclease I, which degrades the unwound 3' tail. The respective recB1080 recQ xonA mutant showed poor viability, DNA repair and homologous recombination deficiency, and very increased illegitimate recombination. These findings demonstrate that the metabolism of the 3' ending overhang is a decisive factor in tuning the balance of homologous and illegitimate recombination in E. coli, thus highlighting the importance of regulating DSB resection for preserving genome integrity. recB mutants used in this study, showing pronounced RecQ helicase and exonuclease I dependence, make up a suitable model system for studying mechanisms of DSB resection in bacteria. Also, these mutants might be useful for investigating functions of the conserved RecQ helicase family members, and congruently serve as a simpler, more defined model system for human oncogenesis.     

RecFOR function is required for DNA repair and recombination in a RecA loading-deficient recB mutant of Escherichia coli

The RecA loading activity of the RecBCD enzyme, together with its helicase and 5' --> 3' exonuclease activities, is essential for recombination in Escherichia coli. One particular mutant in the nuclease catalytic center of RecB, i.e., recB1080, produces an enzyme that does not have nuclease activity and is unable to load RecA protein onto single-stranded DNA. There are, however, previously published contradictory data on the recombination proficiency of this mutant. In a recF(-) background the recB1080 mutant is recombination deficient, whereas in a recF(+) genetic background it is recombination proficient. A possible explanation for these contrasting phenotypes may be that the RecFOR system promotes RecA-single-strand DNA filament formation and replaces the RecA loading defect of the RecB1080CD enzyme. We tested this hypothesis by using three in vivo assays. We compared the recombination proficiencies of recB1080, recO, recR, and recF single mutants and recB1080 recO, recB1080 recR, and recB1080 recF double mutants. We show that RecFOR functions rescue the repair and recombination deficiency of the recB1080 mutant and that RecA loading is independent of RecFOR in the recB1080 recD double mutant where this activity is provided by the RecB1080C(D(-)) enzyme. According to our results as well as previous data, three essential activities for the initiation of recombination in the recB1080 mutant are provided by different proteins, i.e., helicase activity by RecB1080CD, 5' --> 3' exonuclease by RecJ- and RecA-single-stranded DNA filament formation by RecFOR.     

Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding.

Infection of Escherichia coli with phage T4 gene 2am was used to transport 3H-labeled linear duplex DNA into cells to follow its degradation in relation to the cellular genotype. In wild-type cells, 49% of the DNA was made acid soluble within 60 min; in recB or recC cells, only about 5% of the DNA was made acid soluble. Remarkably, in recD cells about 25% of the DNA was rendered acid soluble. The DNA degradation in recD cells depended on intact recB and recC genes. The degradation in recD cells was largely decreased by mutations in recJ (which eliminates the 5' single-strand-specific exonuclease coded by this gene) or xonA (which abolishes the 3' single-strand-specific exonuclease I). In a recD recJ xonA triple mutant, the degradation of linear duplex DNA was roughly at the level of a recB mutant. Results similar to those with the set of recD strains were also obtained with a recC++ mutant (in which the RecD protein is intact but does not function) and its recJ, xonA, and recJ xonA derivatives. The observations provide evidence for a recBC-dependent DNA-unwinding activity that renders unwound DNA susceptible to exonucleolytic degradation. It is proposed that the DNA-unwinding activity causes the efficient recombination, DNA repair, and SOS induction (after application of nalidixic acid) in recD mutants. The RecBC helicase indirectly detected here may have a central function in Chi-dependent recombination and in the recombinational repair of double-strand breaks by the RecBCD pathway.     

Facilitated loading of RecA protein is essential to recombination by RecBCD enzyme

Although the RecB(2109)CD enzyme retains most of the biochemical functions associated with the wild-type RecBCD enzyme, it is completely defective for genetic recombination. Here, we demonstrate that the mutant enzyme exhibits an aberrant double-stranded DNA exonuclease activity, intrinsically producing a 3'-terminal single-stranded DNA overhang that is an ideal substrate for RecA protein-promoted strand invasion. Thus, the mutant enzyme constitutively processes double-stranded DNA in the same manner as the chi-modified wild-type RecBCD enzyme. However, we further show that the RecB(2109)CD enzyme is unable to coordinate the loading of RecA protein onto the single-stranded DNA produced, and we conclude that this inability results in the recombination-defective phenotype of the recB2109 allele. Our findings argue that the facilitated loading of RecA protein by the chi-activated RecBCD enzyme is essential for RecBCD-mediated homologous recombination in vivo.     

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.     

Recombination of bacteriophage lambda in recD mutants of Escherichia coli

RecBCD enzyme is centrally important in homologous recombination in Escherichia coli and is the source of ExoV activity. Null alleles of either the recB or the recC genes, which encode the B and C subunits, respectively, manifest no recombination and none of the nuclease functions characteristic of the holoenzyme. Loss of the D subunit, by a recD mutation, likewise results in loss of ExoV activity. However, mutants lacking the D subunit are competent for homologous recombination. We report that the distribution of exchanges along the chromosome of Red-Gam-phage lambda is strikingly altered by recD null mutations in the host. When lambda DNA replication is blocked, recombination in recD mutant strains is high near lambda's right end. In contrast, recombination in isogenic recD+ strains is approximately uniform along lambda unless the lambda chromosome contains a chi sequence. Recombination in recD mutant strains is focused toward the site of action of a type II restriction enzyme acting in vivo on lambda. The distribution of exchanges in isogenic recD+ strains is scarcely altered by the restriction enzyme (unless the phage contains an otherwise silent chi). The distribution of exchanges in recD mutants is strongly affected by lambda DNA replication. The distribution of exchanges on lambda growing in rec+ cells is not influenced by DNA replication. The exchange distribution along lambda in recD mutant cells is independent of chi in a variety of conditions. Recombination in rec+ cells is chi influenced. Recombination in recD mutants depends on recC function, occurs in strains deleted for rac prophage, and is independent of recJ, which is known to be required for lambda recombination via the RecF pathway. We entertain two models for recombination in recD mutants: (i) recombination in recD mutants may proceed via double-chain break--repair, as it does in lambda's Red pathway and E. coli's RecE pathway; (ii) the RecBC enzyme, missing its D subunit, is equivalent to the wild-type, RecBCD, enzyme after that enzyme has been activated by a chi sequence.     

XthA (Exonuclease III) regulates loading of RecA onto DNA substrates in log phase Escherichia coli cells

Exonucleases can modify DNA substrates created during DNA replication, recombination and repair. In Escherichia coli, the effects of several 3'-5' exonucleases on RecA loading were studied by assaying RecA-GFP foci formation. Mutations in xthA (ExoIII), xseAB (ExoVII), xni (ExoIX), exoX (ExoX) and tatD (ExoXI) increased the number of RecA-GFP foci twofold to threefold in a population of log phase cells grown in minimal medium. These increases depend on xonA. Epistasis analysis shows that ExoVII, ExoX, ExoIX and ExoXI function in a common pathway, distinct from ExoIII (and ExoI is upstream of both pathways). It is shown (paradoxically) that in xthA mutants, RecA-GFP loading is predominantly RecBCD-dependent and that xthA recB double mutants are viable. Experiments show that while log phase xthA cells have twofold more double-stranded breaks (DSBs) than wild type, they do not induce the SOS response. The increase in RecA loading is independent of the base excision repair (BER) proteins Nth, MutM and Nei. It is proposed that log phase cells produce DSBs that do not induce the SOS response. Furthermore, ExoI, ExoIII and the other 3'-5' exonucleases process these DSBs, antagonizing the RecBCD pathway of RecA loading, thus regulating the availability of these substrates for recombination.     

A RecA mutant, RecA(730), suppresses the recombination deficiency of the RecBC(1004)D-chi* interaction in vitro and in vivo

In Escherichia coli, homologous recombination initiated at double-stranded DNA breaks requires the RecBCD enzyme, a multifunctional heterotrimeric complex that possesses processive helicase and exonuclease activities. Upon encountering the DNA regulatory sequence, chi, the enzymatic properties of RecBCD enzyme are altered. Its helicase activity is reduced, the 3'-->5'nuclease activity is attenuated, the 5'-->3' nuclease activity is up-regulated, and it manifests an ability to load RecA protein onto single-stranded DNA. The net result of these changes is the production of a highly recombinogenic structure known as the presynaptic filament. Previously, we found that the recC1004 mutation alters chi-recognition so that this mutant enzyme recognizes an altered chi sequence, chi*, which comprises seven of the original nucleotides in chi, plus four novel nucleotides. Although some consequences of this mutant enzyme-mutant chi interaction could be detected in vivo and in vitro, stimulation of recombination in vivo could not. To resolve this seemingly contradictory observation, we examined the behavior of a RecA mutant, RecA(730), that displays enhanced biochemical activity in vitro and possesses suppressor function in vivo. We show that the recombination deficiency of the RecBC(1004)D-chi* interaction can be overcome by the enhanced ability of RecA(730) to assemble on single-stranded DNA in vitro and in vivo. These data are consistent with findings showing that the loading of RecA protein by RecBCD is necessary in vivo, and they show that RecA proteins with enhanced single-stranded DNA-binding capacity can partially bypass the need for RecBCD-mediated loading.     

Exonuclease requirements for recombination of lambda-phage in recD mutants of Escherichia coli

Recombination of lambda red gam phage in recD mutants is unaffected by inactivation of RecJ exonuclease. Since nucleases play redundant roles in E. coli, we inactivated several exonucleases in a recD mutant and discovered that 5'-3' exonuclease activity of RecJ and exonuclease VII is essential for lambda-recombination, whereas exonucleases of 3'-5' polarity are dispensable. The implications of the presented data on current models for recombination initiation in E. coli are discussed.     

Identification and characterization of recD, a gene affecting plasmid maintenance and recombination in Escherichia coli.

We isolated mutations that reduce plasmid stability in dividing cell populations and mapped these mutations to a previously undescribed gene, recD, that affects recombination frequency and consequently the formation of plasmid concatemers. Insertions of the transposable element Tn10 into recD resulted in increased concatemerization and loss of pSC101 and ColE1-like replicons during nonselective growth. Both concatemer formation and plasmid instability in recD mutants require a functional recA gene. Mutations in recD are recessive to recD+ and map to a small region of the Escherichia coli chromosome located between recB and argA. Although the recD locus is distinct from loci encoding the two previously identified subunits of the RecBC enzyme, mutations in recD appear to affect the exonuclease activity of this enzyme.     

Escherichia coli RecBC pseudorevertants lacking chi recombinational hotspot activity

Pseudorevertants of an Escherichia coli exonuclease V (RecBC enzyme)-negative mutant have been isolated after ethyl methane sulfonate mutagenesis of a recC73 (presumed missense) mutant. The remedial mutations in each of the four pseudorevertants studied in detail map and complement as recC mutations. By several criteria, such as recombination proficiency, support of phage growth, RecBC nuclease activity, and cell viability, the pseudorevertants appear to have regained partially or completely various aspects of RecBC activity. However, chi recombinational hotspots, which stimulate exclusively the RecBC pathway of recombination, have no detectable activity in lambda vegetative crosses in the pseudorevertants. The properties of these mutants, in which the RecBC pathway of recombination is active yet in which chi is not active, are consistent with the hypothesis that wild-type RecBC enzyme directly interacts with chi sites; alternatively, the mutants may block or bypass the productive interaction of another recombinational enzyme with chi.     

Pathways of resistance to thymineless death in Escherichia coli and the function of UvrD

Thymineless death (TLD) is the rapid loss of viability in bacterial, yeast, and human cells starved of thymine. TLD is the mode of action of common anticancer drugs and some antibiotics. TLD in Escherichia coli is accompanied by blocked replication and chromosomal DNA loss and recent work identified activities of recombination protein RecA and the SOS DNA-damage response as causes of TLD. Here, we examine the basis of hypersensitivity to thymine deprivation (hyper-TLD) in mutants that lack the UvrD helicase, which opposes RecA action and participates in some DNA repair mechanisms, RecBCD exonuclease, which degrades double-stranded linear DNA and works with RecA in double-strand-break repair and SOS induction, and RuvABC Holliday-junction resolvase. We report that hyper-TLD in uvrD cells is partly RecA dependent and cannot be attributed to accumulation of intermediates in mismatch repair or nucleotide-excision repair. These data imply that both its known role in opposing RecA and an additional as-yet-unknown function of UvrD promote TLD resistance. The hyper-TLD of ruvABC cells requires RecA but not RecQ or RecJ. The hyper-TLD of recB cells requires neither RecA nor RecQ, implying that neither recombination nor SOS induction causes hyper-TLD in recB cells, and RecQ is not the sole source of double-strand ends (DSEs) during TLD, as previously proposed; models are suggested. These results define pathways by which cells resist TLD and suggest strategies for combating TLD resistance during chemotherapies.     

Effect of a recD mutation on DNA damage resistance and transformation in Deinococcus radiodurans

The bacterium Deinococcus radiodurans is resistant to extremely high levels of DNA-damaging agents such as UV light, ionizing radiation, and chemicals such as hydrogen peroxide and mitomycin C. The organism is able to repair large numbers of double-strand breaks caused by ionizing radiation, in spite of the lack of the RecBCD enzyme, which is essential for double-strand DNA break repair in Escherichia coli and many other bacteria. The D. radiodurans genome sequence indicates that the organism lacks recB and recC genes, but there is a gene encoding a protein with significant similarity to the RecD protein of E. coli and other bacteria. We have generated D. radiodurans strains with a disruption or deletion of the recD gene. The recD mutants are more sensitive than wild-type cells to irradiation with gamma rays and UV light and to treatment with hydrogen peroxide, but they are not sensitive to treatment with mitomycin C and methyl methanesulfonate. The recD mutants also show greater efficiency of transformation by exogenous homologous DNA. These results are the first indication that the D. radiodurans RecD protein has a role in DNA damage repair and/or homologous recombination in the organism.     

Homologous pairing of single-stranded DNA and superhelical double-stranded DNA catalyzed by RecO protein from Escherichia coli.

The recO gene product is required for DNA repair and some types of homologous recombination in wild-type Escherichia coli cells. RecO protein has been previously purified and shown to bind to single- and double-stranded DNA and to promote the renaturation of complementary single-stranded DNA molecules. In this study, purified RecO protein was shown to catalyze the assimilation of single-stranded DNA into homologous superhelical double-stranded DNA, an activity also associated with RecA protein. The RecO protein-promoted strand assimilation reaction requires Mg2+ and is ATP independent. Because of the biochemical similarities between RecO and RecA proteins, the ability of RecO protein to substitute for RecA protein in DNA repair in vivo was also assessed in this study. The results show that overexpression of RecO protein partially suppressed the UV repair deficiency of a recA null mutant and support the hypothesis that RecO and RecA proteins are functionally similar with respect to strand assimilation and the ability to enhance UV survival. These results suggest that RecO and RecA proteins may have common functional properties.     

Exonuclease X of Escherichia coli. A novel 3'-5' DNase and Dnaq superfamily member involved in DNA repair.

DNA exonucleases are critical for DNA replication, repair, and recombination. In the bacterium Escherichia coli there are 14 DNA exonucleases including exonucleases I-IX (including the two DNA polymerase I exonucleases), RecJ exonuclease, SbcCD exonuclease, RNase T, and the exonuclease domains of DNA polymerase II and III. Here we report the discovery and characterization of a new E. coli exonuclease, exonuclease X. Exonuclease X is a member of a superfamily of proteins that have homology to the 3'-5' exonuclease proofreading subunit (DnaQ) of E. coli DNA polymerase III. We have engineered and purified a (His)(6)-exonuclease X fusion protein and characterized its activity. Exonuclease X is a potent distributive exonuclease, capable of degrading both single-stranded and duplex DNA with 3'-5' polarity. Its high affinity for single-strand DNA and its rapid catalytic rate are similar to the processive exonucleases RecJ and exonuclease I. Deletion of the exoX gene exacerbated the UV sensitivity of a strain lacking RecJ, exonuclease I, and exonuclease VII. When overexpressed, exonuclease X is capable of substituting for exonuclease I in UV repair. As we have proposed for the other single-strand DNA exonucleases, exonuclease X may facilitate recombinational repair by pre-synaptic and/or post-synaptic DNA degradation.