Rec A-independent homologous recombination induced by a putative fold-back tetraplex DNA

We have recently reported that a GC-rich palindromic repeat sequence presumably adopts a stable fold-back tetraplex DNA structure under supercoiling. To establish the biological significance of this structure, we inserted this sequence between two direct repeat sequences, separated by 200 bp, in a plasmid. We then investigated the effect of this sequence on homologous recombination events. Here we report that the putative fold-back DNA tetraplex structure induces homologous recombination between direct repeat sequences. Interestingly, this recombination event is independent of recA, a major driving force for homologous recombination. We think that the fold-back structure forces the repeat sequences to come into close proximity and therefore leads to strand exchange. Although triplex-induced recombination has been well documented, our results for the first time directly establish the potential of a tetraplex structure to induce recA-independent homologous recombination in vivo. This finding might have a significant implication for site-directed gene deletion in the context of the correction of genetic defects.     

An<Emphasis Type="Italic"> Arabidopsis</Emphasis> homologue of bacterial RecA that complements an<Emphasis Type="Italic"> E. coli recA</Emphasis> deletion is targeted to plant mitochondria

Homologous recombination results in the exchange and rearrangement of DNA, and thus generates genetic variation in living organisms. RecA is known to function in all bacteria as the central enzyme catalyzing strand transfer and has functional homologues in eukaryotes. Most of our knowledge of homologous recombination in eukaryotes is limited to processes in the nucleus. The mitochondrial genomes of higher plants contain repeated sequences that are known to undergo frequent rearrangements and recombination events. However, very little is known about the proteins involved or the biochemical mechanisms of DNA recombination in plant mitochondria. We provide here the first report of an Arabidopsis thaliana homologue of Escherichia coli RecA that is targeted to mitochondria. The mt recA gene has a putative mitochondrial presequence identified from the A. thaliana genome database. This nuclear gene encodes a predicted product that shows highest sequence homology to chloroplast RecA and RecA proteins from proteobacteria. When fused to the GFP coding sequence, the predicted presequence was able to target the fusion protein to isolated mitochondria but not to chloroplasts. The mitochondrion-specific localization of the mt recA gene product was confirmed by Western analysis using polyclonal antibodies raised against a synthetic peptide from a unique region of the mature mtRecA. The Arabidopsis mt recA gene partially complemented a recA deletion in E. coli, enhancing survival after exposure to DNA-damaging agents. These results suggest a possible role for mt recA in homologous recombination and/or repair in Arabidopsis mitochondria.     

An alternative pathway of recombination of chromosomal fragments precedes recA-dependent recombination in the radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans R1 and other members of this genus are able to repair and survive extreme DNA damage induced by ionizing radiation and many other DNA-damaging agents. The ability of R1 to repair completely > 100 double-strand breaks in its chromosome without lethality or mutagenesis is recA dependent. However, during the first 1.5 h after irradiation, recA+ and recA cells show similar increases in the average size of chromosomal fragments. In recA+ cells, DNA continues to enlarge to wild-type size within 29 h. However, in recA cells, no DNA repair is observed following the first 1.5 h postirradiation. This recA-independent effect was studied further, using two slightly different Escherichia coli plasmids forming adjacent duplication insertions in the chromosome, providing repetitive sequences suitable for circularization by non-recA-dependent pathways following irradiation. After exposure to 1.75 Mrad (17,500 Gy), circular derivatives of the integration units were detected in both recA+ and recA cells. These DNA circles were formed in the first 1.5 h postirradiation, several hours before the onset of detectable recA-dependent homologous recombination. By comparison, D. radiodurans strains containing the same E. coli plasmids as nonrepetitive direct insertions did not form circular derivatives of the integration units before or after irradiation in recA+ or recA cells. The circular derivatives of the tandemly integrated plasmids were formed before the onset of recA-dependent repair and have structures consistent with the hypothesis that DNA repair occurring immediately postirradiation is by a recA-independent single-strand annealing reaction and may be a preparatory step for further DNA repair in wild-type D. radiodurans.     

RecA-independent high-frequency deletion of recombinant cosmid DNA in Escherichia coli

Segments of DNA were deleted from recombinant cosmid DNAs during propagation in Escherichia coli hosts in liquid culture. DNAs of more than 1000 cosmids propagated in various E. coli hosts were analysed by agarose gel electrophoresis (AGE). The effects of vectors, insert DNAs and host genetic characters on the formation of deletions were examined. The probability of deletion and the pattern of deletion bands observed by AGE differed from clone to clone, and after extensive culture the deletion band patterns remained almost constant during further culture. Most recombinant clones eventually showed deletion during prolonged liquid culture. Mutations in the recA gene of E. coli hosts, including a deletion mutation, did not prevent deletion. Most deletions occurred in the insert portions of cosmid DNAs. Nucleotide sequence analysis of six deletion junctions in test cosmid cMB15 demonstrated that deletions occurred between two short complete direct repeats of about 4-10 bp, irrespective of whether the cosmid was propagated in a recA host or a rec+ host. Some deletions occurred at the same sites either in a recA host or a rec+ host. These results suggest that the deletion events are mainly mediated by a recA-independent recombination system(s) of E. coli host cells.     

A physical assay for detection of early meiotic recombination intermediates in Saccharomyces cerevisiae

In most eukaryotic organisms, recombination events leading to exchanges between homologous chromosomes link the homologs in a manner that allows their proper attachment to the meiotic spindle. In the yeast Saccharomyces cerevisiae these exchanges are initiated in early prophase as double-strand breaks in the DNA. These breaks are processed through a series of intermediates to yield mature crossovers late in prophase. The following experiments were designed to monitor the appearance of the earliest recombinant DNA strands formed in this process. A polymerase chain reaction assay was devised that allows the detection of recombinant strands at a known initiation site for meiotic recombination. The time and rate of appearance of recombinant strands was found to coincide with commitment to recombination, demonstrating that DNA strands bearing sequences from both parental chromosomes are rapidly formed after the initiation of meiotic recombination.     

A physical assay for detection of early meiotic recombination intermediates in Saccharomyces cerevisiae

In most eukaryotic organisms, recombination events leading to exchanges between homologous chromosomes link the homologs in a manner that allows their proper attachment to the meiotic spindle. In the yeast Saccharomyces cerevisiae these exchanges are initiated in early prophase as double-strand breaks in the DNA. These breaks are processed through a series of intermediates to yield mature crossovers late in prophase. The following experiments were designed to monitor the appearance of the earliest recombinant DNA strands formed in this process. A polymerase chain reaction assay was devised that allows the detection of recombinant strands at a known initiation site for meiotic recombination. The time and rate of appearance of recombinant strands was found to coincide with commitment to recombination, demonstrating that DNA strands bearing sequences from both parental chromosomes are rapidly formed after the initiation of meiotic recombination. Received: 22 July 1997 / Accepted: 25 February 1998     

Purification and properties of the recA protein of Proteus mirabilis. Comparison with Escherichia coli recA protein; specificity of interaction with single strand binding protein

The product of the cloned recA+ gene of Proteus mirabilis substitutes for a defective recA protein in Escherichia coli recA- mutants and restores recombination, repair, and prophage induction functions to near normal levels (Eitner, G., Adler, B., Lanzov, V. A., and Hofemeister, J. (1982) Mol. Gen. Genet. 185, 481-486). In this paper, we report the purification to near homogeneity of the P. mirabilis recA protein (recApm). The polypeptide has a molecular weight similar to that of E. coli recA protein (recAec) and shows partial identity with recAec when reacted against antibodies specific for the E. coli recA protein. recApm catalyzes the hydrolysis of ATP in the presence of single-stranded but not double-stranded DNA. We have compared the recombination-like activities of recApm with those of recAec and found them to be similar. In the presence of ATP and Mg2+, stoichiometric amounts of recApm promote the complete reciprocal exchange of strands between gapped circular and linear duplex DNA molecules. The enzyme also efficiently promotes the formation of D-loops from circular duplex DNA and homologous single-stranded fragments. However, although recApm and recAec share the above physical and functional similarities, they differ in their ability to interact with the E. coli single strand binding protein to catalyze the transfer of one DNA strand from a linear duplex to a single-stranded circle.     

Degenerate oligonucleotide primers for enzymatic amplification of recA sequences from gram-positive bacteria and mycoplasmas.

RecA protein in gram-negative bacteria, especially in Escherichia coli, has been extensively studied, but little is known about this key enzyme in other procaryotes. Described here are degenerate oligonucleotide primers that have been used to amplify by the polymerase chain reaction (PCR) recA sequences from several gram-positive bacteria and mycoplasmas. The DNA sequences of recA PCR products from Streptococcus pyogenes, Streptococcus mutans, Enterococcus faecalis, and Mycoplasma pulmonis were determined and compared. These data indicate that the M. pulmonis recA gene has diverged significantly from recA genes of other eubacteria. It should be possible to use cloned recA PCR products to construct recA mutants, thereby providing the means of elucidating homologous genetic recombination and DNA repair activities in these organisms.     

Homologous pairing between nucleosome cores on a linear duplex DNA and nucleoprotein filaments of RecA protein-single stranded DNA.

The ability of E coli recA protein to promote homologous pairing with linear duplex DNA bound to HU protein (Nucleosome cores) was found to be differentially affected. The formation of paranemic joint molecules was not affected whereas the formation of plectomic joint molecules was inhibited from the start of the reaction. The formation of paranemic joint molecules between nucleoprotein filaments of recA protein-circular single stranded DNA and closed circular duplex DNA is believed to generate positive supercoiling in the duplex DNA. We found that the positively superhelical duplex DNA was inert in the formation of joint molecules but could be converted into an active substrate, in situ, by the action of wheat germ topoisomerase I. These observations initiate an understanding of the structural features of E coli chromosome such as DNA supercoiling and nucleosome-like structures in homologous recombination.     

Functional structures of the recA protein found by chimera analysis.

We developed a novel genetic method for finding functional regions of a protein by the analysis of chimeras formed between homologous proteins. Sets of chimeric genes were made by intramolecular homologous recombination in a linearized plasmid DNA carrying both recA genes of Escherichia coli and Pseudomonas aeruginosa. A recBCsbcA strain of E. coli was used for isolation of plasmids carrying recombinants between these genes. Examination of properties of E. coli strains deleting the recA gene and carrying a plasmid with a chimeric gene shows that chimera formation at certain positions inactivates a RecA function. Frequently, all chimeras with a junction in a certain region of the protein inactivate a function. Rather than a direct effect of the presence of the junction at a particular position, mismatching of the regions both sides of the junction that are derived from the different species is responsible for the inactivation. For a chimeric protein to be functional, certain pairs of sequences in different regions of the protein must derive from the same parent. Four pairs of such sequences were found: two are involved in activities for genetic recombination and for resistance to ultraviolet light irradiation and the others in formation of active oligomers. Regions defined by these sequences are located in the looped regions of the protein. A pair of regions may co-operate to form a functional folded structure.     

Isolation and Characterization of Escherichia Coli Mutants with Altered Rates of Deletion Formation

Using site-specific mutagenesis in vitro we constructed a genetic system to detect mutants with altered rates of deletion formation between short repeated sequences in Escherichia coli. After in vivo mutagenesis with chemical mutagens and transposons, the system allowed the identification of mutants with either increased or decreased deletion frequencies. One mutational locus, termed mutR, that results in an increase in deletion formation, was studied in detail. The mutR gene maps at 38.5 min on the E. coli genetic map. Since the precise excision of many transposable elements is also mediated at short repeated sequences, we investigated the effects of the mutant alleles, as well as recA, on precise excision of the transposon Tn9. Neither mutR nor recA affect precise excision of the transposon Tn9, from three different insertions in lacI, whereas these alleles do affect other spontaneous deletions in the same system. These results indicate that deletion events leading to precise excision occur principally via a different pathway than other random spontaneous deletions. It is suggested that, whereas precise excision occurs predominantly via a pathway involving replication enzymes (for instance template strand slippage), deletions on an F'factor are stimulated by recombination enzymes.     

Effect of terminal non-homology on intramolecular recombination of linear plasmid substrates in Escherichia coli.

Circular dimer plasmids linearized with a restriction endonuclease undergo intramolecular recombination to yield recombinant circular monomers at high efficiency by a recA-independent mechanism in Escherichia coli recB recC sbcA mutants. The rate of this reaction is at least 1000-fold higher than the recombination rate observed for circular plasmid recombination substrates in the same mutants. Three potential models have been previously proposed to explain the recombination events observed. The validity of these models was tested in recA recB recC sbcA mutants using additional recombination substrates. These substrates, when linearized by incubation with an appropriate restriction enzyme, contain non-homologous adenovirus 2 DNA on one or both ends. The data indicate that terminal non-homology does not significantly affect the efficiency of recovering recombinants. In contrast to many recombination models proposed that involve the invasion of homologous duplex DNA by single-stranded DNA ends, the intramolecular recombination reaction studied here does not appear to involve direct pairing from the end(s) of the substrate DNA. Furthermore, the results are consistent with a model proposing that pairing and strand exchange occur between two homologous duplex regions within the linear dimer molecule.     

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.     

Synthesis of hybrid bacterial plasmids containing highly repeated satellite DNA.

Hybrid plasmid molecules containing tandemly repeated Drosophila satellite DNA were constructed using a modification of the (dA)-(dT) homopolymer procedure of Lobban and Kaiser (1973). Recombinant plasmids recovered after transformation of recA bacteria contained 10% of the amount of satellite DNA present in the transforming molecules. The cloned plasmids were not homogenous in size. Recombinant plasmids isolated from a single colony contained populations of circular molecules which varied both in the length of the satellite region and in the poly(dA)-(dt) regions linking satellite and vector. While subcloning reduced the heterogeneity of these plasmid populations, continued cell growth caused further variations in the size of the repeated regions. Two different simple sequence satellites of Drosophila melanogaster (1.672 and 1.705 g/cm3) were unstable in both recA and recBC hosts and in both pSC101 and pCR1 vectors. We propose that this recA-independent instability of tandemly repeated sequences is due to unequal intramolecular recombination events in replicating DNA molecules, a mechanism analogous to sister chromatid exchange in eucaryotes.     

On the formation of spontaneous deletions: the importance of short sequence homologies in the generation of large deletions

Using lacl-Z fusion strains of Escherichia coli we have devised systems that detect deletions of varying lengths. We examined deletions 700-1000 base pairs long, and genetically characterized over 250 spontaneous deletions. Of these, we analyzed 24 by direct DNA sequencing and 18 by inspection of restriction fragment patterns. Deletions of this size occur almost exclusively at short repeated sequences in both (recA+ and recA- strain backgrounds, but are detected 25-fold more frequently in a recA+ background. The frequency of deletion formation correlates with the extent of homology between the short repeated sequences, although other factors may be involved. The largest hotspot, which accounts for 60% of the deletions detected, involves the largest homology in the system (14 of 17 base pairs). Altering a single base pair within this homology reduces deletion incidence by an order of magnitude. We discuss possible mechanisms of deletion formation and consider its relationship to the excision of transposable elements.     

Branching out: meiotic recombination and its regulation

Homologous recombination is a dynamic process by which DNA sequences and strands are exchanged. In meiosis, the reciprocal DNA recombination events called crossovers are central to the generation of genetic diversity in gametes and are required for homolog segregation in most organisms. Recent studies have shed light on how meiotic crossovers and other recombination products form, how their position and number are regulated and how the DNA molecules undergoing recombination are chosen. These studies indicate that the long-dominant, unifying model of recombination proposed by Szostak et al. applies, with modification, only to a subset of recombination events. Instead, crossover formation and its control involve multiple pathways, with considerable variation among model organisms. These observations force us to 'branch out' in our thinking about meiotic recombination.     

Avoidance of inter-repeat recombination by sequence divergence and a mechanism of neutral evolution

Eucaryotic genomes are loaded with diverse repeated sequences and are therefore threatened by rearrangements via inter-repeat crossovers and by gene-inactivating conversions between genes and their inactive pseudogenes. Such repeated DNA sequences are usually diverged and polymorphic. Sequence divergence by well-spread point mutations is a potent inhibitor of homologous recombination due to the loss of recombination initiation sites and to the editing of recombinational intermediates by the mismatch repair system. Evidence is reviewed suggesting that a germ line process can identify duplicated sequences by homologous pairing, modify them by methylation and mutate by C----T transitions. Since this process requires a minimum contiguous homology that is larger than the average exon size, it is proposed that fragmentation by intron inserts protects the coding sequences from inactivation by homologous interactions with their pseudogene sequences.     

Cloning of the recA gene of Neisseria gonorrhoeae and construction of gonococcal recA mutants.

Interspecific complementation of an Escherichia coli recA mutant was used to identify recombinant plasmids within a genomic cosmid library derived from Neisseria gonorrhoeae that carry the gonococcal recA gene. These plasmids complement the E. coli recA mutation in both homologous recombination functions and resistance to DNA damaging agents. Subcloning, deletion mapping, and transposon Tn5 mutagenesis were used to localize the gonococcal gene responsible for suppression of the E. coli RecA- phenotype. Defined mutations in and near the cloned gonococcal recA gene were constructed in vitro and concurrently associated with a selectable genetic marker for N. gonorrhoeae and the mutated alleles were then reintroduced into the gonococcal chromosome by transformation-mediated marker rescue. This work resulted in the construction of two isogenic strains of N. gonorrhoeae, one of which expresses a reduced proficiency in homologous recombination activity and DNA repair function while the other displays an absolute deficiency in these capacities. These gonococcal mutants behaved similarly to recA mutants of other procaryotic species and displayed phenotypes consistent with the data obtained by heterospecific complementation in an E. coli recA host. The functional activities of the recA gene products of N. gonorrhoeae and E. coli appear to be highly conserved.     

Role of enzymes of homologous recombination in illegitimate plasmid recombination in Bacillus subtilis.

The structural stability of plasmid pGP1, which encodes a fusion between the penicillinase gene (penP) of Bacillus licheniformis and the Escherichia coli lacZ gene, was investigated in Bacillus subtilis strains expressing mutated subunits of the ATP-dependent nuclease, AddAB, and strains lacking the major recombination enzyme, RecA. Strains carrying a mutation in the ATP-binding site of the AddB subunit exhibited high levels of plasmid instability, whereas a comparable mutation in the A subunit did not affect plasmid stability. Using an alternative plasmid system, pGP100, we were able to demonstrate that the differences in stability reflected differences in initial recombination frequencies. Based on a comparison of endpoint sequences observed in the various hosts, we speculate that at least two different mechanisms underlie the deletion events involved, the first (type I) occurring between nonrepeated sequences, and the second (type II) occurring between short direct repeats (DRs). The latter event was independent of single-strand replication intermediates and the mode of replication and possibly requires the introduction of double-strand breaks (DSBs) between the repeats. In the absence of functional AddAB complex, or the AddB subunit, DSBs are likely to be processed via a recA-independent mechanism, resulting in intramolecular recombination between the DRs. In wild-type cells, such DSBs are supposed to be either repaired by a mechanism involving AddAB-dependent recombination or degraded by the AddAB-associated exonuclease activity. Plasmid stability assays in a recA mutant showed that (i) the level of deletion formation was considerably higher in this host and (ii) that deletions between short DRs occurred at higher frequencies than those described previously for the parental strain. We propose that in wild-type cells, the recA gene product is involved in recombinational repair of DSBs.     

Mechanism of intramolecular recyclization and deletion formation following transformation of Escherichia coli with linearized plasmid DNA.

The deletion end-points of a number of type I (less than monomeric) plasmid deletants obtained by transforming recA+ or recA- E. coli with linear pBR322 DNA were determined by DNA sequencing. In both monodirectional and bidirectional deletions the recyclization point was normally characterized by recombination between directly repeated sequences of between 4 and 10 bp present on each arm of the linearized pBR322 molecule. Frequently, short tracts of uninterrupted homology involved in recombinational recircularization were embedded in regions of relative non-homology. A model predicting the probability of matching sequences in either end of a linear plasmid molecule is presented. It is proposed that exonucleolytic processing of the exposed termini of linear plasmid molecules generates substrates for subsequent recombinational recyclization and deletion. The activity of host recombination and repair functions in recircularizing linear DNA molecules explains the generation of many of the aberrant recombinant DNA constructs obtained during gene cloning procedures.