Cloning, Sequencing, and Characterization of the recA Gene from Rhodopseudomonas viridis and Construction of a recA Strain

A recombination-deficient strain of the phototrophic bacterium Rhodopseudomonas viridis was constructed for the homologous expression of modified photosynthetic reaction center genes. The R. viridis recA gene was cloned and subsequently deleted from the R. viridis genome. The cloned R. viridis recA gene shows high identity to known recA genes and was able to complement the Rec⁻ phenotype of a Rhizobium meliloti recA strain. The constructed R. viridis recA strain showed the general Rec⁻ phenotype, i.e., increased sensitivity to DNA damage and severely impaired recombination ability. The latter property of this strain will be of advantage in particular for expression of modified, nonfunctional photosynthetic reaction centers which are not as yet available.     

Involvement of Recombination Genes in Growth and Viability of Escherichia coli K-12

We have studied the growth properties of 17 isogenic strains of Escherichia coli K-12 differing only in the recA, recB, recC, and sbcA alleles. We have observed the following. (i) All recombination deficient strains have decreased growth rates and decreased viabilities compared with recombination proficient strains. The large populations of nonviable cells in Rec⁻ cultures may arise by spontaneous lethal sectoring (9). (ii) A recA mutant strain which is entirely recombination deficient and which shows high ultraviolet sensitivity and “reckless” deoxyribonucleic acid (DNA) breakdown has approximately the same growth rate and twice the viability as recB and recC mutant strains which have residual recombination proficiency, moderate ultraviolet sensitivity, and “cautious” DNA breakdown. (iii) Indirectly suppressed (sbcA⁻) recombination proficient (Rec⁺) revertants of recB and recC mutant strains have approximately normal growth rates and are three times as viable as their Rec⁻ ancestors (but not as viable as rec⁺ cells). We suggest the following hypothesis to account for the low viability of Rec⁻E. coli. Single-strand breaks in the DNA duplex, necessary for normal bacterial growth, may be repaired in a Rec⁺ cell. Failure of Rec⁻ cells to repair this normal DNA damage may lead to the observed loss of viability.     

The effects of mutations in the polA and recA genes on mutagenesis by nitrosoguanidine in Salmonellatyphimurium

Results of semi-quantitative plate tests indicated that polA and recA mutants of Salmonella typhimurium strain LT2 trpB1 might be significantly less mutable by nitrosoguanidine (MNNG) than were their repair-proficient parents strains. Quantitative data obtained in treat-and-plate experiments showed that this was not the case, at least for low doses of MNNG, and also that the recA strain was significantly more mutable at low doses than its Rec⁺ parent. On the basis of these results it is suggested that cells of S. typhimurium may possess a recA⁺-dependent repair pathway capable of error-free removal of MNNG-induced pre-mutational lesions from their DNA.     

Recombination deficient mutant of Caulobacter crescentus

Summary A recombination-deficient (Rec^-) strain of Caulobacter crescentus has been isolated from a collection of mutants sensitive to ultraviolet irradiation. The Rec^- mutant fails to give recombinants following Cr30-mediated generalized transduction or following RP4-mediated conjugation. The recombination frequency in the Rec^- strain is at least 5000-fold lower than in the wild type strains. The Rec^- mutant is indistinguishable from wild type in terms of morphology, growth rate, viability, and phage sensitivities, differing only in properties known to be associated with recA-type mutations in other organisms: recombination frequency, ultraviolet sensitivity, and Weigle reactivation. The map location of the rec-526 allele has not been identified, but rec-526 can be cotransferred with the fla-169 mutation by RP4-mediated conjugation at low frequency. This apparent linkage has been used to move the rec mutation to other strains. The Rec^- mutant resembles recA strains of other organisms and provides a healthy strain severely deficient in recombination for use in complementation and cloning studies involving C. crescentus.     

Isolation of plasmid deletion mutants and study of their instability

We describe a method which allows isolation of deletions within hybrid plasmids. It is based on the fact that the tetracycline resistance (Tc^R) gene of pBR322 can be inactivated by inserting foreign DNA into its HindIII site, and that the easily selectable Tc^R mutants of such plasmids are generally (>90%) due to deletions of certain hybrid plasmid sequences. We have found that Tc^R mutants are usually maintained within the cell recombined with the parental Tc^S plasmids. Such heterodimers dissociate in both Rec⁺ and in recA hosts. Parental rather than mutant plasmids are then retained by the host cell.     

Asymmetric crossing over in the spontaneous formation of large deletions in the tonB-trp region of the Escherichia coli K-12 chromosome

The chromosomal tonB gene of Escherichia coli was used as a target for the detection of spontaneous deletion mutations. The deletions were isolated in both recA ⁺ and recA ^? cells, and mutants carrying large deletions were identified because they also lacked part or all of the trp operon. The frequencies of tonB-trp deletion were 1.79?×?10^?9 and 1.09?×?10^?9 for recA ⁺ and recA ^? cells, respectively. We analyzed 12 deletions from recA ⁺ and 10 from recA ^? cells by cloning and direct sequencing. The deletions ranged in size from 5612?bp to 15142?bp for recA ⁺ and from 5428?bp to 13289 for recA ^? cells. Three deletions from recA ⁺ cells and five deletions from recA ^? cells were found to have occurred between short sequence repeats at the termini of the deletion, leaving one copy of the repeat in the mutant sequence. Seven deletions from recA ⁺ cells and three deletions from recA ^? cells did not have repeats at their termini; in these cases, the DNA sequences that are adjacent to the deletion termini in the wild-type are characterized by short (2–4?bp) repeats. From these results, a model is presented for the generation of deletion mutations which involves formation of an asymmetric crossover mediated by repeated sequences of 2- to 4-bp.     

In vivo loss of supercoiled DNA carrying a palindromic sequence

Summary Interest in the fate of long palindromic DNA sequences in E. coli has been kindled by the observation that their inviability is overcome in recBC sbcB strains and that these hosts permit the construction of DNA libraries containing long palindromic sequences present in the human genome. In this paper we show that a reduction in the level of intracellular supercoiled DNA occurs as the result of the presence of a 530 bp palindrome in bacteriophage lambda. This reduction occurs in Rec⁺ and recA strains but not in strains lacking exonucleases V and I (recBC sbcB). However, the DNA must be active (not repressed) for this reduction to be observed, since it is not seen in a Rec⁺ host lysogenic for phage lambda. These results argue against two hypotheses: firstly, that the palindrome causes inviability solely by interfering with packaging, and secondly, that it dose so solely by interfering with recombination. Conversely, these results suggest that a feature of active monomeric DNA (probably its replication) is involved in inviability.     

Exonuclease VII is involved in “reckless” DNA degradation in UV-irradiated Escherichia coli

The recA mutants of Escherichia coli exhibit an abnormal DNA degradation that starts at sites of double-strand DNA breaks (DSBs), and is mediated by RecBCD exonuclease (ExoV). This “reckless” DNA degradation occurs spontaneously in exponentially growing recA cells, and is stimulated by DNA-damaging agents. We have previously found that the xonA and sbcD mutations, which inactivate exonuclease I (ExoI) and SbcCD nuclease, respectively, markedly suppress “reckless” DNA degradation in UV-irradiated recA cells. In the present work, we show that inactivation of exonuclease VII (ExoVII) by an xseA mutation contributes to attenuation of DNA degradation in UV-irradiated recA mutants. The xseA mutation itself has only a weak effect, however, it acts synergistically with the xonA or sbcD mutations in suppressing “reckless” DNA degradation. The quadruple xseA xonA sbcD recA mutants show no sign of DNA degradation during post-irradiation incubation, suggesting that ExoVII, together with ExoI and SbcCD, plays a crucial role in regulating RecBCD-catalyzed chromosome degradation. We propose that these nucleases act on DSBs to create blunt DNA ends, the preferred substrates for the RecBCD enzyme. In addition, our results show that in UV-irradiated recF recA⁺ cells, the xseA, xonA, and sbcD mutations do not affect RecBCD-mediated DNA repair, suggesting that ExoVII, ExoI and SbcCD nucleases are not essential for the initial targeting of RecBCD to DSBs. It is possible that the DNA-blunting activity provided by ExoVII, ExoI and SbcCD is required for an exchange of RecBCD molecules on dsDNA ends during ongoing “reckless” DNA degradation.     

Emergence of thermostable plasmid pTRZ117 from pTRA117 via temperature-dependent integration and excision is dependent on host property of Rec in Bacillus stearothermophilus

A DNA recombination-deficient Rec⁻ mutant of Bacillus stearothermophilus was obtained via mutagenesis from a parental strain B. stearothermophilus MO-3 deficient in neutral protease npr. The Rec⁻ status was confirmed by the fact that no recombinational revertant appeared when a hybrid plasmid pNP13 carrying npr was used. The performance of the temperature-dependent integrative and excisable plasmid pTRA117 was further studied in this Rec⁻ host. Its integration into host chromosome was found to be dependent on Rec, although flanking-homology integration had been disproved. Consequently, the excision product of pTRA117, a thermostable plasmid pTRZ117, did not emerge in the Rec⁻ host.     

Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli

One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii) in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48 % of all mutations; (iii) in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA^- strain were G:C → T:A transversions, occurring within the sequence which in recA⁺ strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv) in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C → A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations.     

Molecular cloning,sequence and regulation of expression of the recA gene of the phototrophic bacterium Rhodobacter sphaeroides

The recA gene of Rhodobacter sphaeroides 2.4.1 has been isolated by complementation of a UV-sensitive RecA^? mutant of Pseudomonas aeruginosa. Its complete nucleotide sequence consists of 1032 bp, encoding a polypeptide of 343 amino acids. The deduced amino acid sequence displayed highest identity to the RecA proteins from Rhizobium mehloti, Rhizobium phaseoli, and Agrobacterium tumefaciens. An Escherichia coli-like SOS consensus region, which functions as a binding site for the LexA repressor molecule was not present in the 215 by upstream region of the R. sphaeroides recA gene. Nevertheless, by using a recA-lacZ fusion, we have shown that expression of the recA gene of R. sphaeroides is inducible by DNA damage. A recA-defective strain of R. sphaeroides was obtained by replacement of the active recA gene by a gene copy inactived in vitro. The resulting recA mutant exhibited increased sensitivity to UV irradiation, and was impaired in its ability to perform homologous recombination as well as to trigger DNA damage-mediated expression. This is the first recA gene from a Gram-negative bacterium that lacks an E. coli-like SOS box but whose expression has been shown to be DNA damage-inducible and auto-regulated.     

Genetic analysis of constitutive stable DNA replication in rnh mutants of Escherichia coli K12

Summary Escherichia coli rnh mutants deficient in ribonuclease H (RNase H) are capable of DNA replication in the absence of protein synthesis. This constitutive stable DNA replication (SDR) is dependent upon the recA ⁺ gene product. The requirement of SDR for recA ⁺ can be suppressed by rin mutations (for recA⁺-independent), or by lexA(Def) mutations which inactivate the LexA repressor. Thus, there are at least three genetically distinct types of SDR in rnh mutants: recA ⁺-dependent SDR seen in rnh ^- rin⁺ lexA⁺ strains, recA ⁺-independent in rnh ^- rin^- lexA⁺, and recA ⁺-independent in rnh ^- rin⁺ lexA(Def). The expression of SDR in rin ^- and lexA(Def) mutants demonstrated a requirement for RNA synthesis and for the absence of RNase H. The suppression of the recA ⁺ requirement by rin mutations was shown to depend on some new function of the recF ⁺ gene product. In contrast, the suppression by lexA-(Def) mutations was not dependent on recF ⁺. The lexA3 mutation inhibited recA ⁺-dependent SDR via reducing the amount of recA ⁺ activity available, and was suppressed by the recAo254 mutation. The SDR in rnh ^- rin^- cells was also inhibited by the lexA3 mutation, but the inhibition was not reversed by the recAo254 mutation, indicating a requirement for some other lexA ⁺-regulated gene product in the recA ⁺-independent SDR process. A model is presented for the regulation of the expression of these three types of SDR by the products of the lexA ⁺, rin⁺ and recF ⁺ genes.     

Recombination in Escherichia coli. VI. Characterization of a recombination-deficient mutation with unusual properties

A detailed study of the rec-34 mutation shows that this mutation is located near pheA on the E. coli chromosome, like the recA and recH genes. The rec-34 recipients, which are radiation-sensitive and “reckless”, yield about 0.1–1% of the number of recombinants obtained with a Rec⁺ recipient in conjunction experiments. The recombinants obtained in conjunction experiments, in which rec-4⁺ allele transfer is avoided, are still recombination deficient.rec-34 definitely is not a mutation in the recH gene. Although definite proof is lacking we suggest that rec-34 is an unusual recA mutation.     

Single Strand Annealing Plays a Major Role in RecA-Independent Recombination between Repeated Sequences in the Radioresistant Deinococcus radiodurans Bacterium

The bacterium Deinococcus radiodurans is one of the most radioresistant organisms known. It is able to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Our work aims to highlight the genes involved in recombination between 438 bp direct repeats separated by intervening sequences of various lengths ranging from 1,479 bp to 10,500 bp to restore a functional tetA gene in the presence or absence of radiation-induced DNA double strand breaks. The frequency of spontaneous deletion events between the chromosomal direct repeats were the same in recA+ and in ΔrecA, ΔrecF, and ΔrecO bacteria, whereas recombination between chromosomal and plasmid DNA was shown to be strictly dependent on the RecA and RecF proteins. The presence of mutations in one of the repeated sequence reduced, in a MutS-dependent manner, the frequency of the deletion events. The distance between the repeats did not influence the frequencies of deletion events in recA ⁺ as well in ΔrecA bacteria. The absence of the UvrD protein stimulated the recombination between the direct repeats whereas the absence of the DdrB protein, previously shown to be involved in DNA double strand break repair through a single strand annealing (SSA) pathway, strongly reduces the frequency of RecA- (and RecO-) independent deletions events. The absence of the DdrB protein also increased the lethal sectoring of cells devoid of RecA or RecO protein. γ-irradiation of recA ⁺ cells increased about 10-fold the frequencies of the deletion events, but at a lesser extend in cells devoid of the DdrB protein. Altogether, our results suggest a major role of single strand annealing in DNA repeat deletion events in bacteria devoid of the RecA protein, and also in recA ⁺ bacteria exposed to ionizing radiation.     

Some Properties of Excision-defective Recombination-deficient Mutants of Escherichia coli K-12

Strains of Escherichia coli that carry the mutation uvrA6 show no measurable excision of pyrimidine dimers and are easily killed by ultraviolet (UV) light, whereas strains that carry recA13 are defective in genetic recombination and are also UV-sensitive. An Hfr strain carrying uvrA6 was crossed with an F⁻ strain carrying recA13. Among the recombinants identified, one carrying uvrA recA proved to be of exceptional sensitivity to UV light. It is estimated from the UV dose (0.2 erg/mm² at 253.7 nm) required to reduce the number of colony-forming cells by one natural logarithm that about 1.3 pyrimidine dimers were formed in a genome of 5 × 10⁶ base pairs for each lethal event. This double mutant is 40 times more UV-sensitive than the excision-defective strain carrying uvrA6. The replication of one pyrimidine dimer is generally a lethal event in strains carrying recA13. Spontaneous breakdown and UV-induced breakdown of the deoxyribonucleic acid (DNA) of cells of the various genotypes were estimated by growing the cells in medium containing ³H-thymidine and measuring both acid-precipitable and acid-soluble radioactivity. The UV-induced degradation in strains with recA13 did not require the uvr⁺ genes and hence appears to depend upon a mechanism other than dimer excision. The greater level of survival after irradiation in Rec⁺ as compared to Rec⁻ bacteria may be due to a recovery mechanism involving the reconstruction of the bacterial chromosome through genetic exchanges which occur between the newly replicated sister duplexes and which effectively circumvent the damaged bases remaining in the DNA.     

Indirect and intragenic suppression of the lexA102 mutation in E. coli B/r.

Summary In Escherichia coli B/r the expression of UV inducible (SOS) functions is under the control of the recA and lexA genes. In this study we have characterized mutants which are altered in their ability to express SOS functions. These mutants were isolated as UV resistant UV nonmutable (Rnm) derivatives of the lexA102 uvrA155 mutant strain WP51. The UV resistance of these Rnm strains is a result of the suppression of lexA102 mediated UV sensitivity. Genetic mapping of rnm mutations shows that the two predominant classes, rnmA and rnmB, map in or very near the lexA and recA genes respectively. rnmA mutations differ from rnmB with respectively recA protein synthesis. rnmA mutations do not restore the ability to express high levels of recA protein after UV treatment whereas rnmB mutations result in constitutive expression of high levels of recA protein. However, both rnmA and rnmB mutant strains inhibit postirradiation DNA degradation. This shows that in rnmA strains, high levels of recA protein are not needed to inhibit postirradiation DNA degradation.The genetic map location and constitutive expression of recA protein synthesis resulting from rnmB mutations suggests that they are operator constitutive mutations of the recA gene. The result that the lexA ⁺ gene is required for the expression of UV mutagenesis in rnmB mutants shows that high levels of recA protein do not circumvent the need for the lexA ⁺ gene product in this process. Thus, while the lexA gene product is required for the induction of recA protein synthesis, lexA must have an additional role in UV induced mutagenesis.     

The role of recombination in the formation of circular oligomers of the lambda plasmid

The λdv1 plasmid forms an extensive oligomeric series of circular DNA molecules in recombination-proficient (recsu⁺) Escherichia coli. These rec⁺ [λdv1]⁺ strains can be typed into the following four classes according to which member of the oligomeric series is most frequent: monomer, dimer, trimer, and tetramer strains. Each of these strains forms a set of circular λdv1 DNA molecules in which most members belong to the series l, 2l, 3l, 4l, where l is the length of the most frequent circular DNA that characterizes the strain—i.e. l equals the length of the most frequent oligomer in the respective strain. In a given strain, the frequency of a molecular species decreases as its length becomes a larger multiple of l. For example, the dimer strains produce dimers, tetramers, hexamers, octomers, etc., in decreasing frequencies, which reach the limits of detection at about the hexadecamer.When recA^? mutations that are absolutely defective for host recombination are introduced into each of these four strains, l retains the same values as in the parent rec⁺ strain, but oligomers larger than 2l are not formed, and the frequency of the 2l oligomer is much reduced. The introduction of recB^? or recC^? mutations, which are only partially defective for host recombination, produces a much smaller perturbation of the rec⁺ distributions, and $\text{rec}{}^{\mathrm{+}}\text{recA}{}^{\mathrm{?}}$ merodiploids exhibit the rec⁺ phenotype with respect to both oligomerization and host recombination.The effects of rec^? mutations on the distribution of λdv1 oligomers and the nature of the oligomeric series produced in rec⁺ cells all indicate that an intermolecular reciprocal recombination between two circular λdv1 DNAs is the principal reaction responsible for oligomerization. It is suggested that the small residual oligomerization that yields 2l oligomers in recA^?cells results from aberrant segregation of the DNA strands at the termination of the replication of l-sized molecules.The inactivation of recA, but not of recB or C, also results in a marked reduction in the frequency of spontaneous curing which in recA⁺ [λdv1⁺]hosts leads to the segregation of [λdv^?]cells. However, spontaneous curing does not appear to be dependent upon the recombination reactions that yield the [λdv 1⁺]oligomers, since the frequency of oligomerization in recA⁺ hosts decreases with increasing l, whereas the frequency of curing increases with increasing l.