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.     

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

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

The recB gene product is essential for exonuclease V-dependent DNA degradation in vivo

The recB21 mutation abolishes the exonuclease activity of the RecBCD enzyme (exonuclease V) of Escherichia coli. This might be due to the polar effect of recB21 on expression of the recD gene, the product of which is an essential component of the RecBCD enzyme. To achieve synthesis of the recD gene product, the recD+ plasmid was introduced into the recB21 mutant. Degradation of the endogenous DNA damaged by gamma-rays and degradation of the DNA of a phage T4 gene 2 mutant were nevertheless abnormally small in this strain. Thus, the functional recB gene product is required for the degradative function of the RecBCD enzyme.     

Cloning of the recB, recC, and recD genes from Proteus mirabilis in Escherichia coli: in vivo formation of active hybrid enzymes.

We cloned chromosomal DNA fragments from Proteus mirabilis which complement recBCD deletion mutants of Escherichia coli by restoring (i) recombination proficiency in conjugation, (ii) normal resistance to UV irradiation, and (iii) ATP-dependent exonuclease activity for duplex DNA. The data indicate that the order of the genes thyA, recC, recB, recD, and argA is similar in both P. mirabilis and E. coli. Hybrid enzymes formed in vivo were active in repair and recombination.     

Salmonella recD mutations increase recombination in a short sequence transduction assay.

We have identified recD mutants of Salmonella typhimurium by their ability to support growth of phage P22 abc (anti-RecBCD) mutants, whose growth is prevented by normal host RecBCD function. As in Escherichia coli, the recD gene of S. typhimurium lies between the recB and argA genes at min 61 of the genetic map. Plasmids carrying the Salmonella recBCD+ genes restore ATP-dependent exonuclease V activity to an E. coli recBCD deletion mutant. The new Salmonella recD mutations (placed on this plasmid) eliminate the exonuclease activity and enable the plasmid-bearing E. coli deletion mutant to support growth of phage T4 gene 2 mutants. The Salmonella recD mutations caused a 3- to 61-fold increase in the ability of a recipient strain to inherit (by transduction) a large inserted element (MudA prophage; 38 kb). In this cross, recombination events must occur in the short (3-kb) sequences that flank the element in the 44-kb transduced fragment. The effect of the recD mutation depends on the nature of the flanking sequences and is likely to be greatest when those sequences lack a Chi site. The recD mutation appears to minimize fragment degradation and/or cause RecBC-dependent recombination events to occur closer to the ends of the transduced fragment. The effect of a recipient recD mutation was eliminated if the donor P22 phage expressed its Abc (anti-RecBC) function. We hypothesize that in standard (high multiplicity of infection) P22-mediated transduction crosses, recombination is stimulated both by Chi sequences (when present in the transduced fragment) and by the phage-encoded Abc protein which inhibits the host RecBCD exonuclease.     

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.     

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.     

Functions of multiple exonucleases are essential for cell viability, DNA repair and homologous recombination in recD mutants of Escherichia coli

Heterotrimeric RecBCD enzyme unwinds and resects a DNA duplex containing blunt double-stranded ends and directs loading of the strand-exchange protein RecA onto the unwound 3'-ending strand, thereby initiating the majority of recombination in wild-type Escherichia coli. When the enzyme lacks its RecD subunit, the resulting RecBC enzyme, active in recD mutants, is recombination proficient although it has only helicase and RecA loading activity and is not a nuclease. However, E. coli encodes for several other exonucleases that digest double-stranded and single-stranded DNA and thus might act in consort with the RecBC enzyme to efficiently promote recombination reactions. To test this hypothesis, I inactivated multiple exonucleases (i.e., exonuclease I, exonuclease X, exonuclease VII, RecJ, and SbcCD) in recD derivatives of the wild-type and nuclease-deficient recB1067 strain and assessed the ability of the resultant mutants to maintain cell viability and to promote DNA repair and homologous recombination. A complex pattern of overlapping and sometimes competing activities of multiple exonucleases in recD mutants was thus revealed. These exonucleases were shown to be essential for cell viability, DNA repair (of UV- and gamma-induced lesions), and homologous recombination (during Hfr conjugation and P1 transduction), which are dependent on the RecBC enzyme. A model for donor DNA processing in recD transconjugants and transductants was proposed.     

Photoaffinity labeling of rat liver carbamoyl phosphate synthetase I by 8-azido-ATP

8-Azido-ATP has been found to serve as a photoaffinity label for two distinct ATP sites on rat liver carbamoyl phosphate synthetase I and to allow preliminary localization of these sites. In the dark, 8-azido-ATP acted as a competitive inhibitor with respect to ATP. Ultraviolet irradiation of carbamoyl phosphate synthetase I in the presence of 8-azido-ATP led to an irreversible loss of activity. ATP specifically protected against this inactivation. The incorporation of 2 mol of 8-azido-ATP per mol of enzyme was required for complete inactivation. To localize the 8-azido-ATP-binding sites to discrete regions of carbamoyl phosphate synthetase I which appear to be structural domains, the enzyme was photolabeled with [gamma-32P]8-azido-ATP and subjected to limited proteolytic digestion. The resulting model for the functional roles of the domains is that there is one ATP site on each of the two large internal structural domains of the enzyme. Each of these domains was found to contain the consensus sequences A and B common to many other nucleotide-binding proteins (Walker, J.E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951). In addition, there is extensive structural and possibly functional interaction of the smaller N-terminal domain with one of the internal ATP-binding domains, analogous to a subunit interaction observed with the evolutionarily related Escherichia coli carbamoyl phosphate synthetase.     

Photoaffinity cross-linking of the coupling factor 1 from Micrococcus luteus by 3'-arylazido-8-azido-ATP

The vicinity of nucleotide binding sites and the mechanism of ATP synthesis/hydrolysis have been studied with the bifunctional photosensitive ATP analog 3'-arylazido-8-azido-ATP. 3'-Arylazido-8-azido-ATP is hydrolyzed by the F1-ATPase from Micrococcus luteus in the absence of ultraviolet light. Irradiation, by ultraviolet light, of F1-ATPase in the presence of 3'-arylazido-8-azido-ATP results in the specific formation of cross-links between alpha and beta subunits. The results suggest that a hydrolytic nucleotide binding site is located on a beta subunit at or near an alpha subunit, probably at the interface between these subunits. Such a constellation would permit direct subunit-subunit interactions during ATP synthesis/hydrolysis.     

Derivatization of the catalytic subunits of the chloroplast ATPase by 2-azido-ATP and dicyclohexylcarbodiimide. Evidence for catalytically induced interchange of the subunits

Modifications of the catalytic beta subunits of the chloroplast ATPase (CF1-ATPase) are reported which support the proposal that all three subunits participate sequentially during catalysis. The beta subunits of the CF1-ATPase are sufficiently homogeneous to allow detection of their derivatization with dicyclohexylcarbodiimide (DCCD) or the substrate analog 2-azido-ATP by two-dimensional isoelectric focusing. Whether the DCCD reacts with the same beta subunit that tightly binds ATP has not been known. Our results show that when CF1-ATPase is covalently labeled with 2-azido-ATP followed by reaction with DCCD, different beta subunits are labeled. The DCCD labeling does not stop catalytic cooperativity of the CF1-ATPase and allows slow enzyme turnover. When the DCCD-modified enzyme catalyzes 2-azido-ATP cleavage and the enzyme with tightly bound nucleotide is photolyzed, both DCCD-modified and unmodified subunits are randomly labeled by the azido nucleotide. This result is as expected if during the catalytic cycle one beta subunit with unique properties is replaced by another subunit that gains these properties. The participation of all three subunits in the catalytic cycle is suggested by the apparent retention of catalytic cooperativity by the two remaining subunits after one subunit has already catalyzed 2-azido-ATP cleavage and been labeled.     

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.     

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.     

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.     

Alteration by site-directed mutagenesis of the conserved lysine residue in the ATP-binding consensus sequence of the RecD subunit of the Escherichia coli RecBCD enzyme.

The RecD subunit of the RecBCD enzyme from Escherichia coli contains an amino acid sequence common to many enzymes which bind ATP or GTP (Gly-X-X-Gly-X-Gly-Lys-Thr). We have changed the conserved lysine residue (amino acid number 177) in the RecD protein to glutamine to investigate the role of RecD, and ATP-binding to RecD, in the enzymatic activities of RecBCD. The mutant RecD protein assembles with the RecB and RecC subunits and the mutant enzyme, designated RecBCD-K177Q, can be purified in the same way as the wild-type RecBCD enzyme. The mutant RecD subunit in RecBCD-K177Q is photolabeled to a lesser extent by the ATP analogue 8-azido-adenosine-5'-triphosphate than is the wild-type RecD subunit in RecBCD, suggesting that the mutation has reduced the affinity of RecD for ATP.     

Localization of the high-affinity binding site for ATP on the membrane-bound chloroplast ATP synthase

The photoaffinity analog 2-azido-ADP has been used to investigate the high-affinity binding site(s) for ATP on the chloroplast thylakoid membrane. Photophosphorylation of 2-azido-ADP results in the rapid formation of 2-azido-ATP, which remains tightly bound to the membranes after extensive washing. The kinetic parameters of the tight binding of ATP and of 2-azido-ATP are similar (apparent Km = 1-2 microM; maximum extent = 0.2-0.4 nmol/mg of chlorophyll). Ultraviolet irradiation of washed thylakoid membranes containing tightly bound 2-azido-[gamma-32P]ATP induces covalent incorporation of the label exclusively into the beta subunit of the chloroplast coupling factor one. Previous results have shown that the tight binding site for ADP is also located on the beta subunit of the ATP synthase (Czarnecki, J. J., Abbott, M. S., and Selman, B. R. (1983) Eur. J. Biochem. 136, 19-24). To further characterize the tight binding sites for ADP and ATP, the membrane-bound coupling factor has been covalently modified with either tightly bound 2-azido-[gamma-32P]ATP or tightly bound 2-azido-[beta-32P]ADP. The photolabeled beta subunits have been isolated and subjected to partial proteolytic digestion and SDS-gel electrophoresis. The results of these experiments demonstrate that the tight binding sites for ADP and ATP are located on identical portions of beta subunit polypeptide.     

Interaction of lambda Gam protein with the RecD subunit of RecBCD enzyme increases radioresistance of the wild-type Escherichia coli.

By making use of the gam(+)-plasmid, the so-called gam-dependent radioresistance was studied. This resistance is the result of the interaction between Gam protein (encoded by the gam gene of lambda) and RecBCD enzyme of Escherichia coli. gam-dependent radioresistance is observed in recB+ recC+ recD+ but not in recB+ recC+ recD- cells. It is suggested that Gam protein interacts specifically with the RecD subunit of RecBCD enzyme; the RecBC complex probably retains its activity in the presence of this viral protein.     

Effects of lysine-to-glycine mutations in the ATP-binding consensus sequences in the AddA and AddB subunits on the Bacillus subtilis AddAB enzyme activities.

The N-terminal regions of both subunits AddA and AddB of the Bacillus subtilis AddAB enzyme contain amino acid sequences, designated motif I, which are commonly found in ATP-binding enzymes. The functional significance of the motif I regions was studied by replacing the highly conserved lysine residues of the regions in both subunits by glycines and by examination of the resulting mutant enzymes with respect to their enzymatic properties. This study shows that the mutation in subunit AddB hardly affected the ATPase, helicase, and exonuclease activities of the AddAB enzyme. However, the mutation in subunit AddA drastically reduced these activities, as well as the kcat for ATP hydrolysis. The apparent Km for ATP in ATP hydrolysis did not significantly deviate from that of the wild-type enzyme. These results suggest that the lysine residue in motif I of subunit AddA of the AddAB enzyme is not essential for the binding of the nucleotide but has a role in ATP hydrolysis, which is required for the exonuclease and helicase activities of the enzyme.