recC Mutants Research Articles

Stimulation of recombination between homologous sequences on carcinogen-treated plasmid DNA and chromosomal DNA by induction of the SOS response in Escherichia coli K12.

Previous studies have shown that transformation of Escherichia coli by plasmid DNA modified in vitro by carcinogens leads to RecA-dependant recombination between homologous plasmid and chromosomal DNA sequences. The mechanism of this recombination has now been studied using recombination-deficient mutants, and the influence of induction of the SOS response on the level of recombination investigated. Plasmid pNO1523, containing the str+ operon (Sms), has been modified in vitro by either irradiation with UV light, or by reaction with (+/-) trans-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) and used to transform streptomycin-resistant hosts. The formation of Ampr transformants which also carry streptomycin resistance was used as a measure of the level of recombination between plasmid and chromosomal DNA. Transformation of recB and recC mutants produced no change in the level of recombination while in the recF mutant a significant decrease was observed compared to the wild type host. Thermal induction of the SOS response in tif-1 and tif-1 umuC mutants followed by transformation led to a four-fold increase in recombination in both cases. The results suggest that the streptomycin-resistant transformants arise exclusively via a recombinational pathway which is largely dependant on the recF gene product, and that this pathway is influenced by induction of the SOS response. These results are discussed in terms of the mechanism of this recombination.

Effect of ruv mutations on recombination and DNA repair in Escherichia coli K12.

Mutation of the ruv gene of E. coli is associated with sensitivity to radiation, and filamentous growth after transient inhibition of DNA synthesis. The filamentation of ruv strains is abolished by mutations in sfiA or sfiB that prevent SOS induced inhibition of cell division, but this does not restore resistance to UV radiation. Double mutants carrying both ruv and uvr mutations are considerably more sensitive to UV radiation than the single mutants, but there is no additive effect of ruv with recA, recF, recB, or recC mutations. ruv mutations have little effect on conjugal recombination in wild-type strains but confer recombination deficiency and extreme sensitivity to ionizing radiation in recBC sbcB strains. These results, together with the fact that ruv strains are excision proficient and mutable by UV light, are interpreted to suggest that the ruv + product is involved in recombinational repair of damaged DNA rather than in cell division as suggested by Otsuji et al. (1974).

Properties of the RecB and RecC gene products of EscherichiaColi

The properties of the recB and recC gene products of Escherichia coli were studied using recB and recC gene-inserted plasmids. recB mutants and recC mutants lacked ATP-dependent DNase (recBC enzyme) but showed apparent recovery of enzyme activity on introduction of plasmids carrying the recB and recC gene, respectively. The ATP-dependent DNase was also constructed in vitro by mixing the recB and recC gene products encoded by the plasmids with the corresponding gene. Specific labeling of plasmid-encoded proteins by the maxicell method showed that the recB and recC gene products were 135,000 and 125,000 dalton proteins, respectively. These results suggest that the recB and recC genes are the structural genes of the beta and alpha subunits, respectively, of the recBC enzyme. A gene that encodes a protein of about 100,000 daltons was found to be located between the recB and recC genes. But the product of this gene was shown not to be included in the recBC 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.

Plasmidic recombination in Escherichia coli K-12: the role of recF gene function.

Interplasmidic and intraplasmidic recombination proficiencies were determined in E. coli bacterial strains carrying rec mutations. Our results defined the role of recF gene function, recB, recC, and sbcB gene products (exonuclease V and exonuclease I) in plasmidic recombination in wild-type E. coli cells and in cells in which the recE recombination pathway is activated. RecF gene function is required for interplasmidic recombination regardless of the recB recC genotype. Intraplasmidic recombination is recF dependent in cells having a functional exonuclease V, but not in recB recC mutants. Exonuclease V activity inhibits both interplasmidic and intraplasmidic recombination via the recE pathway.

Genetic recombination of bacterial plasmid DNA: Analysis of the effect of recombination-deficient mutations on plasmid recombination

The recombination of two plasmids, pRDK101 and pACYC184, was examined in wild-type Escherichia coli and in various E. coli strains containing different recombination-deficient mutations. Recombination-induced oligomer formation from monomeric plasmids was found to be dependent on the functions of the recA, recB, recC and recF genes. Intramolecular recombination of tetrameric plasmids to form lower-order oligomers and monomers also required the functions of the recA and recF genes but did not require the function of the recB and recC genes. In all cases where recA, recB, recC and recF mutations appeared to block the formation or reduction of plasmid circular oligomers, these effects could be reversed by the presence of an sbcA mutation. Further studies on recombination in vivo were carried out utilizing two tetracycline-sensitive derivatives of the compatible plasmids pACYC184 and pBR322. Recombination events between these two plasmids could be quantitated by measuring the production of tetracycline-resistant cells during the growth of transformants. Plasmid recombination was found to be reduced by recA and recF mutations, and the effect of these two mutations was reversed by the presence of an sbcA mutation. Plasmid recombination measured by this genetic assay was stimulated by recB recC mutations, and electrophoretic analysis of the recombination products demonstrated that they were primarily circular monomers. A role for the recB recC gene product, exonuclease V, in the resolution of recombination intermediates is discussed.

Construction of recombinant lambda phages that carry the E. coli recB and recC genes.

A fragment of the E. coli chromosome including the recC gene has been cloned by in vitro recombinant DNA techniques into a phage lambda vector to give the recombinant phage lambda drecC. This was used to derive the phage lambda drecBC by in vivo recombination. On lysogenisation of recB and recC mutants with lambda drecBC wild levels of UV-resistance and RecBC DNase activity were restored. Infection of E coli with lambda drecBC led to the synthesis of phage-coded proteins of 125 kilodaltons (kd) and 135 kd that were not synthesised on infection with the original lambda vector, whereas a 125 kd protein but not a 135 kd protein was synthesised in similar experiments with lambda drecC. The recombinant phages, which are unable to form plaques, presumably due to the deletion of essential phage genes during their construction, provided useful starting points from which to subclone the recB, recC, and the neighbouring thyA and argA genes individually into multiple copy plasmid vectors.

RecA-independent general genetic recombination of plasmids.

The small circular genomes of several bacteriophages and a number of plasmids have been used extensively to study various aspects of the mechanism of genetic recombination. Several studies have demonstrated that the interconversion between the circular monomers and larger circular oligomers of many plasmid DNAs is mediated, at least in part, by the pathway which carries out genetic recombination in wild-type Escherichia coli1–4. The recA, recB and recC mutations block conjugation-mediated recombination in E. coli5,6. However, Clark and his co-workers7,8 isolated two mutations, sbcA and sbcB, which indirectly suppress the recombination deficiency of recB and recC mutations. These indirect suppressor mutations seem to cause recombination by other pathways which require the recA gene product as well as products of other genes such as recF, recJ and recM, which are not normally required for recombination in E. coli9,10. Here we report an investigation of the effect of recombination-deficient mutations on plasmid recombination in E. coli. We show that sbcA mutations induce a new plasmid recombination pathway that is 10–15 times more efficient than the normal wild-type recombination pathway and independent of the recA and recF functions.

Induction of lambdoid prophages by amino acid deprivation: differential inducibility; role of recA.

Lambda prophage in auxotrophic lysogens can be induced by omission of one or combinations of the required amino acids from the culture medium. Such amino acid deprivation can result in nearly as effective induction of lambda as thymine deprivation. Prophage 424 is also induced equally effectively under both conditions although to a lesser extent than lambda. By contrast prophage 21 and lambda i21 are differentially induced effectively by thymine deprivation and virtually not at all during amino acid deprivation. The same differential induction of 21 and equivalent induction of lambda and 424 occur when all three prophages are present in the same lysogen. Increasing the levels of lambda repressor with a cI carrying-plasmid prevented amino acidless induction of lambda as did the lambda ind- mutation. A recA, but not a recB, mutation in the host prevented induction by amino acid deprivation. A recC mutant host showed increased spontaneous induction of lambda and 21 prophages. The findings reported are used as an argument that the recA protease probably is not itself acting as the inducing protease and that a likely source of the observed specificity is an effector molecule. Different effector molecules may be produced in response to different exigent situations, to which the phage repressors may have evolved sensitivity. lambda i80 was inducible both by amino acid and thymine deprivation.

Plasmid control of recombination of E. coli K12.

The recombination proficiency of three recipient strains of Escherichia coli K12 carrying different plasmids was investigated by conjugal mating with Hfr Cavalli. Some plasmids (e.g. R1drd 19, R6K) caused a marked reduction in the yield of recombinants formed in crosses with Hfr but did not reduce the ability of host strains to accept plasmid F104. The effect of plasmids on recombination was host-dependent. In Hfr crosses with AB1157 (R1-19) used as a recipient the linkage between selected and unselected proximal markers of the donor was sharply decreased. Plasmid R1-19 also decreased the yield of recombinants formed by recF, recL, and recB recC sbcA mutants, showed no effect on the recombination proficiency of recB recC sbcB mutant, and increased the recombination proficiency of recB, recB recC sbcB recF, and recB recC sbcB recL mutants. An ATP-dependent exonuclease activity was found in all tested recB recC mutants carrying plasmid R1-19, while this plasmid did not affect the activity of exonuclease I in strain AB1157 and its rec- derivatives. The same plasmid was also found to protect different rec- derivatives of the strain AB1157 against the lethal action of UV light. We suppose that a new ATP-dependent exonuclease determined by R1-19 plays a role in both repair and recombination of the host through the substitution of or competition with the exoV coded for by the genes recB and recC.

Pyrimidine dimer excision in Escherichia coli strains deficient in exonucleases V and VII and in the 5' leads to 3' exonuclease of DNA polymerase I

An isogenic series of Escherichia coli strains deficient in various combinations of three 5' leads to 3' exonucleases (exonuclease V, exonuclease VII, and the 5' leads to 3' exonuclease of DNA polymerase I) was constructed and examined for the ability to excise pyrimidine dimers after UV irradiation. Although the recB and recC mutations (deficient in exonuclease V) proved to be incompatible with the polA(Ex) mutation (deficient in the 5' leads to 3' exonuclease of DNA polymerase I), it was possible to reduce the level of the recB,C exonuclease by the use of temperature-sensitive recB270 recC271 mutants. It was found that, by employing strains deficient in exonuclease V, postirradiation DNA degradation could be reduced and dimer excision measurements could be facilitated. Mutants deficient in exonuclease V were found to excise dimers at a rate comparable to that of the wild type. Mutants deficient in exonuclease V and the 5' leads to 3' exonuclease of DNA polymerase I are slightly slower than the wild type at removing dimers accumulated after doses in excess of 40 J/m2. However, although strains with reduced levels of exonuclease VII excised dimers at the same rate as the wild type, the addition of an exonuclease VII deficiency to a strain with reduced levels of exonuclease V and the 5' leads to 3' exonuclease of DNA polymerase I caused a marked decrease in the rate and extent of dimer excision. These observations support previous indications that the 5' leads to 3' exonuclease of DNA polymerase I is important in dimer removal and also suggest a role for exonuclease VII in the excision repair process.

Suppressibility of recA, recB, and recC mutations by nonsense suppressors

Mutations in the recA, recB, and recC genes of Escherichia coli K-12 were surveyed to ascertain whether or not they are suppressed by nonsense suppressors. Several mutations which map in or near the recA gene, but have not been called recA mutations, were also surveyed. An amber recB mutation, recB156, and an amber recC mutation, recC155, were isolated. One recB mutation, recB95, and four recC mutations, recC22, recC38, recC82, and recC83, were found to be suppressed by a UGA suppressor. In addition to the previously isolated amber recA mutation recA99, two other recA mutations, recA52 and recA123, were found to be suppressed by amber suppressor supD32 but not by supE44.

Carcinogen-induced DNA repair in nucleotide-permeable Escherichia coli cells. Analysis of DNA repair induced by the carcinogens N-acetoxy-N-2-acetylaminofluorene and 7-bromomethyl-benz(a)anthracene.

Upon exposure to the carcinogens N-acetoxy-N-2-acetylaminofluorene and 7-bromomethyl-benz[a]anthracene, which bind covalently to DNA, ether-permeabilized (nucleotide-permeable) Escherichia coli wild-type cells responded with DNA excision repair. This repair was missing in mutants carrying defects in genes uvrA, uvrB and uvrC, whereas it was present in uvrD and several rec mutants. Enzymic activities involved were identified by measuring repair polymerization and size reduction of denatured DNA. 1. An easily measurable effect in E. coli wild-type cells was carcinogen-induced repair polymerization. When initiated by N-acetoxy-N-2-acetylaminofluorene or 7-bromomethyl-benz[a]anthracene, it depended upon an ATP-requiring step; CTP, GTP or UTP did not substitute for ATP. DNA repair synthesis was inhibited by p-chloromercuribenzoate and quinacrine. In uvrA, uvrB and uvrC mutants no carcinogen-stimulated DNA synthesis could be detected, indicating that steps involved in pyrimidine dimer excision are also involved in chemorepair. In recA, recB and recC mutant cells, repair synthesis was stimulated by the carcinogens to a normal extent. This evidence excludes the ATP-dependent recB,C deoxyribonuclease and recA gene products as playing an important role in carcinogen-induced excision repair. polA1 cells showed drastically reduced levels of rapair polymerization, indicating that DNA polymerase I is the main polymerizing enzyme. 2. As determined by DNA size reduction in alkaline sucrose gradients, the arylalkylating carcinogens caused endonucleolytic cleavage of endogenous DNA in wild-type cells. This incision step was most effectively performed in the presence of ATP; UTP, CTP and GTP were only slightly effective. Incision was inhibited by p-chloromercuribenzoate and quinacrine. When exposed to the arylalkylating carcinogens, uvrA, uvrB and uvrC mutant cells did not perform the incision step in the presence of ATP, suggesting the involvement of the respective gene products in the initiation of chemorepair.

Analysis of temperature-sensitive recB and recC mutations.

The in vivo pleiotropic effects associated with the temperature-sensitive recB270 and recC271 mutations have been correlated with the in vitro behavior of the recBC nucleases coded for by these alleles. The ATP-dependent breakdown of double-stranded DNA is essential for cell viability, radiation repair, and genetic recombination. Temperature sensitivity can be suppressed in vitro and in vivo.

Interaction of the exrA mutation with rec mutants of Escherichia coli K 12

Mutants of Escherichia coli K 12 were constructed which carry the exrA mutation addition to the various recombination deficient mutations recA recB and recC. The double mutant containing the exrA recA genotype is found to be slightly more sensitive to UV irradiation at very low doses of UV but essentially is very similar to the exrA + recA at the high UV doses. The recombination deficiency, λ induction and DNA degradation of the exrA recA shows a slight increase in the defectiveness of each of these functions. The double mutants of exrA recB and exrA recC show an increase in UV sensitivity and recombination deficiency and λ induction. The DNA degradation following UV-irradiation of these mutants is more characteristic of the recB and recC mutant alone. These results give further support to the theory that exrA and lex are probably mutants within the same cistron and also suggest that exrA, lex and recA are involved in a common DNA repair pathway and that the gene products of all three functions are required to regulate recB+ and recC+ endonuclease induced DNA degradation.

Differential thermolability of exonuclease and endonuclease activities of the recBC nuclease isolated from thermosensitive recB and recC mutants.

The recBC nuclease (also called exonuclease V) has been partially purified from Escherichia coli K-12 strains carrying the thermosensitive recB270, recC271, and recB270 recC271 mutations. Of the multiple activities associated with the enzyme, only the adenosine 5'-triphosphate-dependent exonucleolytic hydrolysis of duplex deoxyribonucleic acid (DNA) is abnormally thermolabile. The exo- and endonucleolytic degradation of single-stranded DNA is no more thermosensitive than that catalyzed by the wild-type enzyme. These results suggest that the defects in genetic recombination, DNA repair, and the maintenance of cell viability observed in recBC mutants in vivo result primarily from the specific loss of adenosine 5'-triphosphate-dependent exonuclease active on duplex DNA.

The recBC deoxyribonuclease of Escherichia coli: isolation and characterization of the subunit proteins and reconstitution of the enzyme.

After dissociation of the E. coli recBc DNase (ATP-dependent DNase) with concentrated NaCl, two subunit proteins were isolated by ion exchange chromatography. Combination and subsequent incubation of the subunits resulted in the appearance of the original DNase. The subunit proteins, designated alpha and beta, have s(20,omega) of 4.1 S and 8.1 S, respectively. The alpha subunit possesses neither the ATP-dependent Dnase nor the DNA-dependent ATPase of the original enzyme. The beta subunit contains a low level of both enzymatic activities in a ratio markedly different from that of the original enzyme. The beta subunit complemented extracts from both recB and recC mutant strains to produce recBC DNase, while the alpha subunit did not complement either extract. These results suggest that recB and recC genes are both required for the production of beta subunit and that the recBC DNase molecule contains a protein component (alpha) that is not determined by either the recB or the recC gene.

The genetic location of the sbcA gene of Escherichia coli.

sbcA, a gene defined by mutations which restore recombination proficiency to RecB and RecC mutants, has been located at minute 29 of the E. coli genetic map. Two independent sbcA mutations have been mapped in this location.

Some properties of mutants of Escherichia coli K12 defective in DNA polymerase I and Exonuclease V

The double mutant polA1 recB21 appears to be viable, although it grows poorly. By contrast, double mutants of either recB21 or recC22 with the temperature sensitive mutation polA12 are inviable at non-permissive temperatures. This suggests that some function of DNA polymerase I, the polA+ gene product, is more deficient in polA12 cells at the nonpermissive temperature than it is in polA1 cells. Differences between polA12 and polA1 mutations are also evident when ultraviolet light and γ-ray survival curves of polA1 recB21 strains are compared with those of polA12 recB21 and polA12 recC22 at various temperatures. Unlike polA1 recB21 double mutants in which the sensitivity associated with each mutation is not additive polA12 recB21 and polA12 recC22 double mutants are approximately twice as radiation sensitive as recB21 and recC22 single mutants at 37°. Both polA12 recB21 and polA12 recC22 mutants grow normally at 41° if they acquire an sbcA suppressor mutation. This mutation results in the appearance of a new deoxyribonuclease which can apparently substitute for the Exonuclease V missing in recB and recC mutants. The viability of the polA1 recB21 double mutant does not appear to be due to the presence of suppressors of either mutation. However, this strain grows much better if it, too, acquires an sbcA suppressor mutation.

Ultraviolet mutagenesis in repair-deficient derivatives of Escherichia coli B/r: uvrA uvrB and uvrA recC strains

UV mutability of uvrA derivatives of E. coli B/r carrying recB or recC mutations was compared with that of otherwise isogenic Rec + strains. The frequency of UV-induced mutations per survivor from Try − to Try + and from Str-s to Str-r, as well as the absolute yields of these mutations, was significantly lower in the recB and recC strains thatn in Rec + at the same UV doses. The results support the hypothesis that UV-induced mutations in these strains originate as errors associated with the occurrence of recA +-dependent post-replication repair of gaps in DNA opposite pyrimidine dimers, and that maximal UV mutability requires participation of the recB- recC nuclease in this repair process. The reduced mutability of recB and recC strains may reflect a change in the accuracy of post-replication repair, such that fewer errors are made per gap repaired than in Rec + strains. Alternatively, the accuracy of post-replication repair may be unchanged, and the reduced UV mutability may result from the reduced tolerance of recB and recC strains for exercised pyrimidine dimers, with consequent selective survival of a fraction of the irradiated poplation containing fewer dimers per cell, compared to the surviving fraction in Rec + at the same UV dose. Data obtained at very low doses of UV indicate that reduced dimer tolerance alone cannot account for the observed reduction in UV mutability of recB and recC strains, unless it is assumed that dimer tolerance is not equally reduced in different regions of the genome.