Genetic and Physical Analysis of Plasmid Recombination in Recb Recc Sbcb and Recb Recc Sbca Escherichia Coli K-12 Mutants

The effect of mutations in known recombination genes (recA, recB, recC, recE, recF, recJ, recN, recO, recQ and ruv) on intramolecular recombination of plasmids was studied in recB recC sbcB and recB recC sbcA Escherichia coli mutants. The rate of recombination of circular dimer plasmids was at least 1000-fold higher in recB recC sbcB or recB recC sbcA mutants as compared to wild-type cells. The rate was decreased by mutations in recA, recF, recJ, recO, ruv or mutS in recB recC sbcB mutants, and by mutations in recE, recN, recO, recQ, ruv or mutS in recB recC sbcA mutants. In addition to measuring the recombination rate of circular dimer plasmids, the recombination-mediated transformation of linear dimer plasmids was also studied. Linear dimer plasmids transformed recB recC sbcB and recB recC sbcA mutants 20- to 40-fold more efficiently than wild-type cells. The transformation efficiency of linear dimer plasmids in recB recC sbcB mutants was decreased by mutations in recA, recF, recJ, recO, recQ or lexA (lexA3). In recB recC sbcA mutants the transformation efficiency of linear dimers was decreased only by a recE mutation. Physical analysis of linear dimer- or circular dimer-transformed recB recC sbcB mutants revealed that all transformants contained recombinant monomer genotypes. This suggests that recombination in recB recC sbcB cells is very efficient.     

The recombination genes addAB are not restricted to gram-positive bacteria: genetic analysis of the recombination initiation enzymes RecF and AddAB in Rhizobium etli

Single-strand gaps (SSGs) and double-strand breaks (DSBs) are the major initiation sites for recombination. In bacteria, the SSGs are repaired by RecFOR, while the DSBs are processed by RecBCD in gram-negative bacteria and AddAB in gram-positive bacteria. Unexpectedly, instead of recBCD genes, the addAB genes were found in members of the alpha-proteobacteria group (gram negative). Taking Rhizobium etli as a model, the role of recF and addAB genes in homologous recombination and repair of damaged DNA was evaluated. Inactivation of either recF or addA provoked strong sensitivity to UV radiation and mitomycin C, while an additive effect was observed in the recF-addA mutant. The DSBs generated by nalidixic acid caused low viability only in the addA mutant. The recombination frequency of large and small plasmids was reduced in the recF mutant (24- and 36-fold, respectively), whereas a slight decrease (threefold) in the addA mutant was observed. Moreover, an additive effect (47- and 90-fold, respectively) was observed in the double mutant, but it was not as dramatic as that in a recA mutant. Interestingly, the frequency of deletion and Campbell-type recombination was slightly affected in either single or double mutants. These results suggest that another pathway exists that allows plasmid and Campbell-type recombination in the absence of recF and addA genes.     

Genetic recombination of bacterial plasmid DNA. Physical and genetic analysis of the products of plasmid recombination in Escherichia coli

Derivatives of plasmid pBR322 DNA containing tet mutations were constructed by inserting XhoI linkers at various sites in the tetracycline resistance gene. Monomer plasmids containing either the tet-10 allele located at nucleotide position 23 or the tet-14 allele located at nucleotide position 1267 were used to construct a circular dimer containing one copy of each allele and a circular trimer containing one copy of the tet-10 allele and two copies of the tet-14 allele. Genetic recombination of these plasmid DNAs to produce a functional tetracycline resistance gene could be detected as the production of tetracycline-resistant progeny during the growth of transformants or using a restriction mapping assay which detected the rearrangement of the mutant alleles. The structure of individual tetracycline-resistant recombination products was determined by restriction mapping. This analysis suggested that as many as 70% of the plasmid recombination events in Escherichia coli AB1157 could have involved gene conversion events. The formation of these recombination products was most easily predicted by a model involving figure 8 recombination intermediates and the formation of symmetric regions of heteroduplex. Recombination in JC10287 delta(srlR-recA)304 occurred at 5% of the wild-type frequency and appeared to occur by a similar mechanism. Recombination in JC9604 recA56 recB21 recC22 sbcA23 occurred at 20 times the wild-type frequency and appeared to involve multiple independent recombination events.     

Concatemer formation of ColE1-type plasmids in mutants of Escherichia coli lacking RNase H activity

rnh mutants harboring pBR322 were found to contain several slowly migrating DNA species when examined by agarose gel electrophoresis. The plasmid DNA from rnh mutants included large molecules, i.e. plasmids two, three or four times the size of a single plasmid unit. That this DNA contained concatemeric plasmid joined in a head-to-tail fashion was determined by digestion with restriction endonucleases that cleaved the monomeric plasmid DNA at a unique site. This treatment resulted in migration of the plasmid DNA at a mobility identical to that of linearized monomeric plasmid by agarose gel electrophoresis. This was confirmed by electron microscopy. Plasmid concatemer formation was detected with several high-copy-number (relaxed type) plasmids but not with low-copy-number (stringent) plasmids. Concatemer formation was dependent on RecA+ and RecF+ functions since several recA and recF mutations abolished concatemer formation. ColE1-type plasmids were previously shown to replicate in rnh mutants in the absence of DNA polymerase I (PolI) activity. This DNA PolI-independent plasmid replication was also examined for its dependence on the recF and recA gene products. rnh- polA(Ts) recF- strains were efficiently transformed with these plasmids at 30 degrees C and 42 degrees C, indicating the presence of DNA PolI-independent replication under recF- conditions. The presence or absence of plasmid replication in rnh- polA- recA(Ts) strains was also examined by measuring the increase in total amounts of plasmid. The results indicated that DNA PolI-independent replication occurred in these triple mutants at 42 degrees C as well as at 30 degrees C. It was concluded that the recombination event giving rise to concatemer formation was not essential for DNA PolI-independent replication in rnh mutants.     

Homologous recombination and mutagenesis of gamma-irradiated plasmid DNA in Escherichia coli host cells

Plasmid DNA was used to study gamma-radiation-induced recombination and mutagenesis in Escherichia coli host cells. Plasmid pBRP1, a derivative of pBR322 containing the lac operon of E. coli, was irradiated with 60Co gamma rays prior to transformation into E. coli strains of different recA and lac genotypes. Plasmid-chromosome recombination was assayed in lacY1 host cells, whereas plasmid mutagenesis was assayed in delta lac host cells lacking chromosomal sequences homologous to the plasmid. Both recombinant and mutant plasmids were identified by the phenotypic changes in lactose utilization, and confirmed by restriction analysis of isolated plasmids. Plasmid-chromosome recombination was induced to high levels (about 20% of survivors at 700 Gy) and was dependent on the host recA gene. Plasmid mutagenesis occurred at lower levels (about 1.5% of survivors at 600 Gy) and was relatively independent of the recA gene. Plasmid survival was unaffected by the presence or absence of host recA mutations or the potential for plasmid-chromosome recombination.     

UV-mutagenesis in Bacillus subtilis. VII. Mutability of recA, recB and recF strains]

UV-light induces reversions to threonine-independence in BD170 Rec+ cells, and does not induce them in isogenic strain BD241 recF. Reversions to methionine-independence are induced in cells GSY1028 recB, and are not in isogenic strain GSY1025 recA. It is possible to construct, with the use of transformation, a viable strain carrying mutations recF and su, the latter imparts threinine-independence to cells. Hence, the absence of UV-induced reversions in recF (and probably in recA) cells can not be explained by the lethal effect of joining, in one genome, two mutations each of which decreases a viability of bacteria. Formation of UV-induced forward mutations which provide additional growth requirements to bacteria is not disturbed in strains recA and recF. Experimental data allow to conclude that UV-induced mutagenesis is not a function of any of two known mechanisms of recombination which function in Bacillus subtilis cells.     

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.     

Suppression of recA deficiency in plasmid recombination by bacteriophage lambda beta protein in RecBCD- ExoI- Escherichia coli cells.

Plasmid recombination, like other homologous recombination in Escherichia coli, requires RecA protein in most conditions. We have found that the plasmid recombination defect in a recA mutant can be efficiently suppressed by the beta protein of bacteriophage lambda. beta protein is required for homologous recombination of lambda chromosomes during lytic phage growth in a recA host and is known to have a strand-annealing activity resembling that of RecA protein. The bioluminescence recombination assay was used for genetic analysis of beta-protein-mediated plasmid recombination. Efficient suppression of the recA mutation by beta protein required the absence of the E. coli nucleases exonuclease I and RecBCD nuclease. These nucleases inhibit a RecA-mediated plasmid recombination pathway that is more efficient than the pathway functioning in wild-type cells. Like RecA-mediated plasmid recombination in RecBCD- ExoI- cells, beta-protein-mediated plasmid recombination depended on concurrent DNA replication and on the activity of the recQ gene. However, unlike RecA-mediated plasmid recombination, beta-protein-mediated recombination in RecBCD- ExoI- cells was independent of recF and recJ activities. We propose that inactivation of exonuclease I and RecBCD nuclease stabilizes a recombination intermediate that is involved in RecA- and beta-protein-catalyzed homologous pairing reactions. We suggest that the intermediate may be linear plasmid DNA with a protruding 3' end, since these nucleases are known to interfere with the synthesis of such linear forms. The different recF and recJ requirements for beta-protein-dependent and RecA-dependent recombinations imply that the mechanisms of formation or processing of the putative intermediate differ in the two cases.     

RecOR suppression of recF mutant phenotypes in Escherichia coli K-12.

The recF, recO, and recR genes form the recFOR epistasis group for DNA repair. recF mutants are sensitive to UV irradiation and fail to properly induce the SOS response. Using plasmid derivatives that overexpress combinations of the recO+ and recR+ genes, we tested the hypothesis that high-level expression of recO+ and recR+ (recOR) in vivo will indirectly suppress the recF mutant phenotypes mentioned above. We found that overexpression of just recR+ from the plasmid will partially suppress both phenotypes. Expression of the chromosomal recO+ gene is essential for the recR+ suppression. Hence we call this RecOR suppression of recF mutant phenotypes. RecOR suppression of SOS induction is more efficient with recO+ expression from a plasmid than with recO+ expression from the chromosome. This is not true for RecOR suppression of UV sensitivity (the two are equal). Comparison of RecOR suppression with the suppression caused by recA801 and recA803 shows that RecOR suppression of UV sensitivity is more effective than recA803 suppression and that RecOR suppression of UV sensitivity, like recA801 suppression, requires recJ+. We present a model that explains the data and proposes a function for the recFOR epistasis group in the induction of the SOS response and recombinational DNA repair.     

Homologous Recombination in ESCHERICHIA COLI: Dependence on Substrate Length and Homology

We studied the in vivo recombination between homologous DNA sequences cloned in phage lambda and a pBR322-derived plasmid by assaying for the formation of phage-plasmid cointegrates by a single (or an odd number of) reciprocal exchange. (1) Recombination proceeds by the RecBC pathway in wild-type cells and by low levels of a RecF-dependent pathway in recBC- cells. The RecE pathway appears not to generate phage-plasmid cointegrates. (2) Recombination is linearly dependent on the length of the homologous sequences. In both RecBC and RecF-dependent pathways there is a minimal length, called the minimal efficient processing segment (MEPS), below which recombination becomes inefficient. The length of MEPS is between 23-27 base pairs (bp) and between 44-90 bp for the RecBC- and RecF-dependent pathways, respectively. A model, based on overlapping MEPS, of the correlation of genetic length with physical length is presented. The bases for the different MEPS length of the two pathways are discussed in relationship to the enzymes specific to each pathway. (3) The RecBC and the RecF-dependent pathways are each very sensitive to substrate homology. In wild-type E. coli, reduction of homology from 100% to 90% decreases recombinant frequency over 40-fold. The homology dependence of the RecBC and RecF-dependent pathways are similar. This suggests that a component common to both, probably recA, is responsible for the recognition of homology.     

Suppression of the UV-sensitive phenotype of Escherichia coli recF mutants by recA(Srf) and recA(Tif) mutations requires recJ+.

Mutations in recA, such as recA801(Srf) (suppressor of RecF) or recA441(Tif) (temperature-induced filamentation) partially suppress the deficiency in postreplication repair of UV damage conferred by recF mutations. We observed that spontaneous recA(Srf) mutants accumulated in cultures of recB recC sbcB sulA::Mu dX(Ap lac) lexA51 recF cells because they grew faster than the parental strain. We show that in a uvrA recB+ recC+ genetic background there are two prerequisites for the suppression by recA(Srf) of the UV-sensitive phenotype of recF mutants. (i) The recA(Srf) protein must be provided in increased amounts either by SOS derepression or by a recA operator-constitutive mutation in a lexA(Ind) (no induction of SOS functions) genetic background. (ii) The gene recJ, which has been shown previously to be involved in the recF pathway of recombination and repair, must be functional. The level of expression of recJ in a lexA(Ind) strain suffices for full suppression. Suppression by recA441 at 30 degrees C also depends on recJ+. The hampered induction by UV of the SOS gene uvrA seen in a recF mutant was improved by a recA(Srf) mutation. This improvement did not require recJ+. We suggest that recA(Srf) and recA(Tif) mutant proteins can operate in postreplication repair independent of recF by using the recJ+ function.     

Effects of the Escherichia coli recF suppressor mutation, recA801, on recF-dependent DNA-repair associated phenomena

In recb recC sbcB mutants genetic recombination is dependent upon the recF gene. recA801, recA802 and recA803 (formerly called srfA mutations) were originally isolated as mutations that suppress recombination deficiency caused by a recF mutation in a recB recC sbcB genetic background. Since the recA801 mutation also suppressed some of the UV sensitivity due to recF143, we sought to determine what DNA-repair pathways were actually being restored by the recA801 mutation in this genetic background. In this paper we show that the suppression of recF143 by recA801 does not extend to the recF143-mediated defects in induced repair of UV-damaged phages. In addition, we show that recA801 suppresses only slightly the recF143-associated defect in induced expression of the SOS-regulated muc genes of pKM101. These results suggest that recA801 suppresses primarily the RecF pathway of recombinational repair.     

Intra- and intermolecular recombination of test plasmids in K12 Escherichia coli cells carrying an RTF derivative of the R1drd-19 plasmid

The RTF derivative of the plasmid R1drd-19 was found to stimulate recombination of the tester plasmids in a recB mutant of Escherichia coli K12. The frequency of intramolecular recombination is increased 3.5 and 20-fold, as compared to the one in rec+ and rec- strains, respectively. The frequency of interplasmid recombination is enhanced 4 and 9-fold, respectively. Considerable heterogeneity of the recombination products of the tester plasmid intramolecular recombination in recB-/RTFR1-19 strain has been revealed. It is hypothesized that a "recombinase" encoded by Rldrd-19 plasmid determines a new minor pathway in recB- (Rec P) which differs in activity and, perhaps substrate specificity from the main Rec BCD pathway.     

The Mechanism of Reca Pola Lethality: Suppression by Reca-Independent Recombination Repair Activated by the Lexa(def) Mutation in Escherichia Coli

The mechanism of recA polA lethality in Escherichia coli has been studied. Complementation tests have indicated that both the 5' -> 3' exonuclease and the polymerization activities of DNA polymerase I are essential for viability in the absence of RecA protein, whereas the viability and DNA replication of DNA polymerase I-defective cells depend on the recombinase activity of RecA. An alkaline sucrose gradient sedimentation analysis has indicated that RecA has only a minor role in Okazaki fragment processing. Double-strand break repair is proposed for the major role of RecA in the absence of DNA polymerase I. The lexA(Def)::Tn5 mutation has previously been shown to suppress the temperature-sensitive growth of recA200(Ts) polA25::spc mutants. The lexA(Def) mutation can alleviate impaired DNA synthesis in the recA200(Ts) polA25::spc mutant cells at the restrictive temperature. recF(+) is essential for this suppression pathway. recJ and recQ mutations have minor but significant adverse effects on the suppression. The recA200(Ts) allele in the recA200(Ts) polA25::spc lexA(Def) mutant can be replaced by δrecA, indicating that the lexA(Def)-induced suppression is RecA independent. lexA(Def) reduces the sensitivity of δrecA polA25::spc cells to UV damage by ~10(4)-fold. lexA(Def) also restores P1 transduction proficiency to the δrecA polA25::spc mutant to a level that is 7.3% of the recA(+) wild type. These results suggest that lexA(Def) activates a RecA-independent, RecF-dependent recombination repair pathway that suppresses the defect in DNA replication in recA polA double mutants.     

Recombination is essential for viability of an Escherichia coli dam (DNA adenine methyltransferase) mutant

Double mutants of Escherichia coli dam (DNA adenine methyltransferase) strains with ruvA, ruvB, or ruvC could not be constructed, whereas dam derivatives with recD, recF, recJ, and recR were viable. The ruv gene products are required for Holliday junction translocation and resolution of recombination intermediates. A dam recG (Holliday junction translocation) mutant strain was isolated but at a very much lower frequency than expected. The inviability of a dam lexA (Ind(-)) host was abrogated by the simultaneous presence of plasmids encoding both recA and ruvAB. This result indicates that of more than 20 SOS genes, only recA and ruvAB need to be derepressed to allow for dam mutant survival. The presence of mutS or mutL mutations allowed the construction of dam lexA (Ind(-)) derivatives. The requirement for recA, recB, recC, ruvA, ruvB, ruvC, and possibly recG gene expression indicates that recombination is essential for viability of dam bacteria probably to repair DNA double-strand breaks. The effect of mutS and mutL mutations indicates that DNA mismatch repair is the ultimate source of most of these DNA breaks. The requirement for recombination also suggests an explanation for the sensitivity of dam cells to certain DNA-damaging agents.     

Plasmid recombination in Haemophilus influenzae

DNA recombination in exponential phase and competent Haemophilus influenzae was measured by an electron microscopic assay that relies on the conversion of plasmid RSF0885 monomers into multimeric forms. Dimer circles were present at a frequency of 2% in plasmid preparations from competent Rd (wild-type) cells; multimers were present at a frequency of 0.2% in preparations from exponential phase cells. Thus, plasmid recombination was stimulated in competent cells. Multimer formation occurred efficiently in cells of the transformation defective mutant rec2, implying that the rec2 gene product is not required for plasmid recombination. However, the absence of multimer plasmids in preparations from competent cells of the transformation defective mutant rec1 suggests that the rec1 gene product is required. Digestion of purified plasmids with restriction endonuclease PvuII, which makes a single cut in the monomer, revealed the presence of recombination intermediates composed of two linear plasmids joined to form two pairs of arms resembling the Greek letter chi. Length measurements of these arms taken from a population of recombination intermediates gave evidence that the plasmids were joined at sites of homology. The distributions of individual DNA strands, at the intersections of the four arms, could be resolved in some recombination intermediates and were of two types. The first type of junction appeared as a single-stranded square with one double-stranded arm appended to each corner. The second type of junction consisted of a single strand of DNA linking the two linear plasmids at a site of homology. The single-stranded linker was frequently situated at the edge of a short gap on one of the plasmids in the pair. The fine structures of the recombinational joints have been interpreted in terms of previously proposed models of recombination.     

Genetic transformation in bacteria

Certain species of bacteria can become competent to take up high molecular weight DNA from the surrounding medium. DNA homologous to resident chromosomal DNA is transported, processed and recombined with the resident DNA. There are some variations in steps leading to transformation between Gram-positive bacteria likebiplococcus pneumoniae and Gram-negative bacteria represented byHaemophilus influenzae but the integration is by single-strand displacement in both cases. Plasmid (RSF0885) transformation is low inHaemophilus influenzae but this is increased significantly if (homologous) chromosomal DNA is spliced to plasmid DNA. InHaemophilus influenzae, rec1 function is required for peak transformation with chimeric plasmids. Chimeric plasmid fixed presumably extrachromosomally undergoes frequent recombination between homologous segments contained in resident chromosome and the plasmid.     

Postreplication DNA repair in Escherichia coli cells. III. Repair independent of the presence of pKM101 and COLIb-P9 plasmids

The presence of pKM101 or ColIb-P9 plasmids in E. coli leads to the increase in the survival of UV-irradiated cells of wild type and of polAI, recB21 recC22 and dnaGts mutants; it does not change the survival of recA13 and lex3 mutants and does not influence kinetics and efficiency of postreplication repair (PRR) of DNA in cells of all the strains examined (with the exception of PG3 dnaGts mutant whose PRR of DNA in the presence of pKM101 plasmid is somewhat lower). The survival of both plasmid-containing and plasmid-free bacteria treated with chloramphenicol decreases in the same degree, but the survival of chloramphenicol-treated recA13, lex3 recB21 rec C22 mutants does not change. The pKM101 plasmid does not lend the dnaGts mutant a new capacity of repairing postreplication gaps with the participation of inducible component of PRR; the chloramphenicol-sensitive component of PRR is absent in this mutant. Plasmid and plasmid-free E. coli strains of wild type and of the polA1 mutant do not differ by the kinetics and level of inducible chloramphenicol-sensitive component of PRR of DNA.     

Homologous Recombination Involving a Large Heterology in Escherichia Coli

Recombination between two different deletion alleles of a gene (neo) for neomycin and kanamycin resistance was studied in an Escherichia coli sbcA- recB-C- strain. The two homologous regions were in an inverted orientation on the same plasmid molecule. Kanamycin-resistant plasmids were selected and analyzed. The rate of recombination to form kanamycin-resistant plasmids was decreased by mutations in the recE, recF and recJ genes, but was not decreased by a mutation in the recA gene. It was found that these plasmids often possessed one wild-type kanamycin-resistant allele (neo+) while the other neo allele was still in its original (deletion) form. Among kanamycin-resistant plasmids with one wild-type and one parental allele it was often found that the region between the inverted repeats had been flipped (turned around) with respect to sites outside the inverted repeats. These results were interpreted as follows. Gene conversion, analogous to gene conversion in eukaryotic meiosis, is responsible for a unidirectional transfer of information from one neo deletion allele to the other. The flipping of the region between the inverted repeats is interpreted as analogous to the crossing over associated with gene conversion in eukaryotic meiosis. In contrast with a rec+ strain, these products cannot be explained by two rounds of reciprocal crossing over involving a dimeric form as an intermediate. In the accompanying paper we present evidence that gene conversion by double-strand gap repair takes place in the same E. coli strain.