RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium

Expression of the bacterial RecA protein in plants stimulates homologous recombination in tobacco. Here we show that RecA plays a direct role in DNA strand exchange in vivo. The number of sister chromatid exchanges (SCEs) was increased 2.4-fold over wild type in transgenic tobacco plants expressing a nuclear-targeted RecA (nt-RecA) protein and could not be increased further by DNA damage, which caused a doubling of the baseline SCE frequency in wild-type plants. Although gene targeting requires homologous recombination, the number of targeted gene replacements was not increased markedly by the presence of nt-RecA by using Agrobacterium-mediated transformation. However, the number of double-strand breaks that were repaired at both sides by homologous recombination was increased 3.3-fold. Stimulation of SCE and fidelity of double-strand break repair by nt-RecA, but not by gene targeting, suggests that the stimulatory activity of RecA is linked to active DNA synthesis. Therefore, nascent replication-associated single strands may be a prerequisite for RecA action in plant cells.     

The synapsis event in the homologous pairing of DNAs: RecA recognizes and pairs less than one helical repeat of DNA.

A key step in homologous recombination is the alignment and pairing of homologous DNAs. The Escherichia coli RecA protein initiates pairing by binding to single-strand DNA, forming a helical nucleoprotein filament. We demonstrate that in the presence of the nonhydrolyzable ATP analogue adenosine 5'-[gamma-thio]triphosphate and ADP, RecA can pair a homologous oligonucleotide 15 bases long with a duplex DNA to yield synaptic complexes consisting of the oligonucleotide and duplex DNA stabilized by RecA. RecA can pair as few as eight bases of homology to form such synaptic complexes. The homologous DNAs remain paired to each other upon removal of RecA provided that the length of shared homology is at least 26 base pairs. Based on our findings and the work of others, we propose that in vitro, one helical turn of a RecA nucleoprotein filament containing approximately six RecA monomers and 15 bases of single-strand DNA is the functional unit sufficient to carry out the homology search.     

The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA

The Holliday junction (HJ), a cross-shaped structure that physically links the two DNA helices, is a key intermediate in homologous recombination, DNA repair, and replication. Several helicase-like proteins are known to bind HJs and promote their branch migration (BM) by translocating along DNA at the expense of ATP hydrolysis. Surprisingly, the bacterial recombinase protein RecA and its eukaryotic homologue Rad51 also promote BM of HJs despite the fact they do not bind HJs preferentially and do not translocate along DNA. RecA/Rad51 plays a key role in DNA double-stranded break repair and homologous recombination. RecA/Rad51 binds to ssDNA and forms contiguous filaments that promote the search for homologous DNA sequences and DNA strand exchange. The mechanism of BM promoted by RecA/RAD51 is unknown. Here, we demonstrate that cycles of RecA/Rad51 polymerization and dissociation coupled with ATP hydrolysis drives the BM of HJs.     

The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair

The RecBCD enzyme is required for homologous recombination and DNA repair in Escherichia coli. The structure and function of RecBCD enzyme is altered on its interaction with the recombination hotspot Chi (5'-GCTGGTGG-3'). It has been hypothesized that the RecD subunit plays a role in Chi-dependent regulation of enzyme activity [Thaler, D. S., Sampson, E., Siddiqi, I., Rosenberg, S. M., Stahl, F. W. & Stahl, M. (1988) in Mechanisms and Consequences of DNA Damage Processing, eds. Friedberg, E. & Hanawalt, P. (Liss, New York), pp. 413-422; Churchill, J. J., Anderson, D. G. & Kowalczykowski, S. C. (1999) Genes Dev. 13, 901-911]. We tested the hypothesis that the RecD subunit inhibits recombination by deleting recD from the nuclease- and recombination-deficient mutant recB(D1080A)CD. We report here that the resulting strain, recB(D1080A)C, was proficient for recombination and DNA repair. Recombination proficiency was accompanied by a change in enzyme activity: RecB(D1080A)C enzyme loaded RecA protein onto DNA during DNA unwinding whereas RecB(D1080A)CD enzyme did not. Together, these genetic and biochemical results demonstrate that RecA loading by RecBCD enzyme is required for recombination in E. coli cells and suggest that RecD interferes with the enzyme domain required for its loading. A nuclease-dependent signal appears to be required for a change in RecD that allows RecA loading. Because RecA loading is not observed with wild-type RecBCD enzyme until it acts at a Chi site, our observations support the view that RecD inhibits recombination until the enzyme acts at Chi.     

A Holliday junction resolvase from Pyrococcus furiosus: functional similarity to Escherichia coli RuvC provides evidence for conserved mechanism of homologous recombination in Bacteria, Eukarya, and Archaea.

The Holliday junction is an essential intermediate of homologous recombination. RecA of Bacteria, Rad51 of Eukarya, and RadA of Archaea are structural and functional homologs. These proteins play a pivotal role in the formation of Holliday junctions from two homologous DNA duplexes. RuvC is a specific endonuclease that resolves Holliday junctions in Bacteria. A Holliday junction-resolving activity has been found in both yeast and mammalian cells. To examine whether the paradigm of homologous recombination apply to Archaea, we assayed and found the activity to resolve a synthetic Holliday junction in crude extract of Pyrococcus furiosus cells. The gene, hjc (Holliday junction cleavage), encodes a protein composed of 123 amino acids, whose sequence is not similar to that of any proteins with known function. However, all four archaea, whose total genome sequences have been published, have the homologous genes. The purified Hjc protein cleaved the recombination intermediates formed by RecA in vitro. These results support the notion that the formation and resolution of Holliday junction is the common mechanism of homologous recombination in the three domains of life.     

Human Dmc1 protein binds DNA as an octameric ring

The bacterial RecA protein has been the most intensively studied enzyme in homologous genetic recombination. The core of RecA is structurally homologous to that of the F1-ATPase and helicases. Like the F1-ATPase and ring helicases, RecA forms a hexameric ring. The human Dmc1 (hDmc1) protein, a meiosis-specific recombinase, is homologous to RecA. We show that hDmc1 forms octameric rings. Unlike RecA and Rad51, however, hDmc1 protein does not form helical filaments. The hDmc1 ring binds DNA in the central channel, as do the ring helicases, which is likely to represent the active form of the protein. These observations indicate that the conservation of the RecA-like ring structure extends from bacteria to humans, and that some RecA homologs may form both rings and filaments, whereas others may function only as rings.     

A homolog of Escherichia coli RecA protein in plastids of higher plants.

Studies of chloroplast DNA variations, and several direct experimental observations, indicate the existence of recombination ability in algal and higher plant plastids. However, no studies have been done of the biochemical pathways involved. Using a part of a cyanobacterial recA gene as a probe in Southern blots, we have found homologous sequences in total DNA from Pisum sativum and Arabidopsis thaliana and in a cDNA library from Arabidopsis. A cDNA was cloned and sequenced, and its predicted amino acid sequence is 60.7% identical to that of the cyanobacterial RecA protein. This finding is consistent with our other results showing both DNA strand transfer activity and the existence of a protein of the predicted molecular mass crossreactive with antibodies to Escherichia coli RecA in the stroma of pea chloroplasts.     

Spontaneous mitotic homologous recombination at an enhanced yellow fluorescent protein (EYFP) cDNA direct repeat in transgenic mice

A transgenic mouse has been created that provides a powerful tool for revealing genetic and environmental factors that modulate mitotic homologous recombination. The fluorescent yellow direct-repeat (FYDR) mice described here carry two different copies of expression cassettes for truncated coding sequences of the enhanced yellow fluorescent protein (EYFP), arranged in tandem. Homologous recombination between these repeated elements can restore full-length EYFP coding sequence to yield a fluorescent phenotype, and the resulting fluorescent recombinant cells are rapidly quantifiable by flow cytometry. Analysis of genomic DNA from recombined FYDR cells shows that this mouse model detects gene conversions, and based on the arrangement of the integrated recombination substrate, unequal sister-chromatid exchanges and repair of collapsed replication forks are also expected to reconstitute EYFP coding sequence. The rate of spontaneous recombination in primary fibroblasts derived from adult ear tissue is 1.3 +/- 0.1 per 106 cell divisions. Interestingly, the rate is approximately 10-fold greater in fibroblasts derived from embryonic tissue. We observe an approximately 15-fold increase in the frequency of recombinant cells in cultures of ear fibroblasts when exposed to mitomycin C, which is consistent with the ability of interstrand crosslinks to induce homologous recombination. In addition to studies of recombination in cultured primary cells, the frequency of recombinant cells present in skin was also measured by direct analysis of disaggregated cells. Thus, the FYDR mouse model can be used for studies of mitotic homologous recombination both in vitro and in vivo.     

Stress-induced intrachromosomal recombination in plant somatic cells

Levels of induced homologous recombination between chromosomal repeats in plant somatic cells were examined. Transgenic plants of Nicotiana tabacum hemi- or homozygous for pairs of deletion derivatives of the neomycin phosphotransferase (nptII) marker gene integrated at a single genomic locus were produced. Homologous recombination within the overlapping parts of the nptII gene restored the function and the resulting kanamycin resistance was used for scoring recombination frequency. The recombination events were confirmed by the appearance of a characteristic 1245-base-pair EcoRV fragment detected in all kanamycin-resistant clones tested. The rate of spontaneous recombination was found to be related to the copy number of recombination substrates and was 9 x 10(-5) and 19 x 10(-5) for hemi- and homozygote strains, respectively. Ionizing radiation, mitomycin C, and heat shock markedly increased the frequency of intrachromosomal recombination. Low doses of x-rays (1.25 Gy) enhanced the relative recombination frequency to approximately twice the spontaneous value. The presence of mitomycin C increased the frequency of recombination 9-fold and exposure to an elevated temperature (50 degrees C) increased it 6.5-fold. The x-ray and heat shock treatments reduced cell viability to 53% and 8%, respectively. Mitomycin C treatment had no effect on cell survival.     

Substoichiometric shifting in the plant mitochondrial genome is influenced by a gene homologous to MutS

The plant mitochondrial genome is retained in a multipartite structure that arises by a process of repeat-mediated homologous recombination. Low-frequency ectopic recombination also occurs, often producing sequence chimeras, aberrant ORFs, and novel subgenomic DNA molecules. This genomic plasticity may distinguish the plant mitochondrion from mammalian and fungal types. In plants, relative copy number of recombination-derived subgenomic DNA molecules within mitochondria is controlled by nuclear genes, and a genomic shifting process can result in their differential copy number suppression to nearly undetectable levels. We have cloned a nuclear gene that regulates mitochondrial substoichiometric shifting in Arabidopsis. The CHM gene was shown to encode a protein related to the MutS protein of Escherichia coli that is involved in mismatch repair and DNA recombination. We postulate that the process of substoichiometric shifting in plants may be a consequence of ectopic recombination suppression or replication stalling at ectopic recombination sites to effect molecule-specific copy number modulation.     

Inhibition of homologous recombination by variants of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs)

Two major DNA double-strand break repair pathways exist in all eukaryotes, nonhomologous DNA end joining (NHEJ) and homologous recombination (HR). Although both pathways can function throughout the cell cycle, NHEJ predominates in G0/G1) (when a replicated sister chromatid is unavailable), whereas HR makes a more substantial contribution in S and G2. How a cell chooses between these two important DNA repair pathways is largely unknown. DNA-dependent protein kinase (DNA-PK) is critical for NHEJ. Here, we describe two conserved splice variants of a catalytic subunit of DNA-PK (DNA-PKcs) that are expressed predominately in nondividing cells. Although both encode stable products, neither reverses the NHEJ defects in DNA-PKcs-deficient cells. In fact, cells expressing one of the DNA-PKcs variants are slightly more radiosensitive than cells completely deficient in DNA-PKcs. We investigated whether cells expressing the DNA-PKcs variants had any other DNA repair deficits and found that these cells are considerably more sensitive to both etoposide and mitomycin C than cells that express no DNA-PKcs at all. Because repair of DNA damage induced by these two agents requires intact HR, we tested whether the NHEJ-defective variants of DNA-PKcs inhibit double-strand break-induced HR in an integrated substrate. In cells expressing the NHEJ-defective variants, HR was markedly reduced. Because the splice variants are expressed highly only in nondividing cells, quiescent cells would be afforded a mechanism to inhibit repair by means of HR when sister chromatids are not available as templates for accurate repair with low risk of genome rearrangement, thereby enhancing genome stability.     

Resolution of an early RecA-recombination intermediate by a junction-specific endonuclease

The nucleoprotein filament formed on a circular single strand by Escherichia coli RecA protein in vitro can pair with homologous duplex DNA even when the latter lacks a free homologous end, but subsequent progression of the reaction through strand exchange requires an end in at least one strand of the duplex DNA. We purified from E. coli an endonuclease activity that cleaves the outgoing strand of duplex DNA at the junction of homologous and heterologous sequences in three-stranded RecA-recombination intermediates. This endonuclease activity also cleaves specifically at the junctions of duplex and single-stranded regions in synthetic double-stranded oligonucleotides whose central portion consists of unpaired heterologous sequences. These activities are consistent with a role in recombination and repair of DNA.     

RecBCD-dependent joint molecule formation promoted by the Escherichia coli RecA and SSB proteins.

We describe the formation of homologously paired joint molecules in an in vitro reaction that is dependent on the concerted actions of purified RecA and RecBCD proteins and is stimulated by single-stranded DNA-binding protein (SSB). RecBCD enzyme initiates the process by unwinding the linear double-stranded DNA to produce single-stranded DNA, which is trapped by SSB and RecA. RecA uses this single-stranded DNA to catalyze the invasion of a supercoiled double-stranded DNA molecule, forming a homologously paired joint molecule. At low RecBCD enzyme concentrations, the rate-limiting step is the unwinding of duplex DNA by RecBCD, whereas at higher RecBCD concentrations, the rate-limiting step is RecA-catalyzed strand invasion. The behavior of mutant RecA proteins in this in vitro reaction parallels their in vivo phenotypes, suggesting that this reaction may define biochemical steps that occur during homologous recombination by the RecBCD pathway in vivo.     

SOS induction and autoregulation of the himA gene for site-specific recombination in Escherichia coli.

The himA gene of EScherichia coli controls the lysogenization of bacteriophage lambda at the level of catalysis of site-specific recombination and expression of the lambda int and cI genes required for lysogenic development. We have analyzed the regulation of himA by two methods: (i) beta-galactosidase synthesis from a lacZ gene inserted into the himA gene and (ii) detection of radioactive HimA protein after fractionation by two-dimensional gel electrophoresis. We find that himA- mutations produce enhanced expression of the himA gene, indicating that HimA protein controls its own synthesis. The himA gene is also induced by treatment of cells with UV or mitomycin C, suggesting control by the inducible DNA repair (SOS) system regulated by the LexA and RecA proteins. Regulation of himA follows the pattern expected for a typical SOS gene: constitutive high expression in mutants that have inactive LexA or the altered RecA conferred by the recA441 (tif1) mutation and low noninducible expression in a mutant that has a deleted recA gene. We conclude that the himA gene is a component of the inducible SoS response, repressed by LexA and induced by the capacity of activated RecA to cleave LexA. We suggest that HimA may be subject to SOS induction because it functions as an "acquisitionase" for new genetic material and thus is of special utility under conditions of impaired capacity for growth of the bacterial population.     

Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: A possible advantage of DNA over RNA as genomic material

Heteroduplex joints are general intermediates of homologous genetic recombination in DNA genomes. A heteroduplex joint is formed between a single-stranded region (or tail), derived from a cleaved parental double-stranded DNA, and homologous regions in another parental double-stranded DNA, in a reaction mediated by the RecA/Rad51-family of proteins. In this reaction, a RecA/Rad51-family protein first forms a filamentous complex with the single-stranded DNA, and then interacts with the double-stranded DNA in a search for homology. Studies of the three-dimensional structures of single-stranded DNA bound either to Escherichia coli RecA or Saccharomyces cerevisiae Rad51 have revealed a novel extended DNA structure. This structure contains a hydrophobic interaction between the 2' methylene moiety of each deoxyribose and the aromatic ring of the following base, which allows bases to rotate horizontally through the interconversion of sugar puckers. This base rotation explains the mechanism of the homology search and base-pair switch between double-stranded and single-stranded DNA during the formation of heteroduplex joints. The pivotal role of the 2' methylene-base interaction in the heteroduplex joint formation is supported by comparing the recombination of RNA genomes with that of DNA genomes. Some simple organisms with DNA genomes induce homologous recombination when they encounter conditions that are unfavorable for their survival. The extended DNA structure confers a dynamic property on the otherwise chemically and genetically stable double-stranded DNA, enabling gene segment rearrangements without disturbing the coding frame (i.e., protein-segment shuffling). These properties may give an extensive evolutionary advantage to DNA.     

Polarity of DNA strand exchange promoted by recombination proteins of the RecA family

Homologs of Escherichia coli RecA recombination protein, which have been found throughout the living kingdom, promote homologous pairing and strand exchange. The nucleoprotein filament, within which strand exchange occurs, has been conserved through evolution, but conservation of the polarity of exchange and the significance of that directionality has not been settled. Using oligonucleotides as substrates, and assays based on fluorescence resonance energy transfer (FRET), we distinguished the biased formation of homologous joints at either end of duplex DNA from the subsequent directionality of strand exchange. As with E. coli RecA protein, the homologous Rad51 proteins from both Homo sapiens (HsRad51) and Saccharomyces cerevisiae (ScRad51) propagated DNA strand exchange preferentially in the 5′ to 3′ direction. The data suggest that 5′ to 3′ polarity is a conserved intrinsic property of recombination filaments.     

Integration of foreign DNA during natural transformation of Acinetobacter sp. by homology-facilitated illegitimate recombination

The active uptake of extracellular DNA and its genomic integration is termed natural transformation and constitutes a major horizontal gene-transfer mechanism in prokaryotes. Chromosomal DNA transferred within a species can be integrated effectively by homologous recombination, whereas foreign DNA with low or no sequence homology would rely on illegitimate recombination events, which are rare. By using the nptII(+) gene (kanamycin resistance) as selectable marker, we found that the integration of foreign DNA into the genome of the Gram-negative Acinetobacter sp. BD413 during transformation indeed was at least 10(9)-fold lower than that of homologous DNA. However, integration of foreign DNA increased at least 10(5)-fold when it was linked on one side to a piece of DNA homologous to the recipient genome. Analysis of foreign DNA integration sites revealed short stretches of sequence identity (3-8 bp) between donor and recipient DNA, indicating illegitimate recombination events. These findings suggest that homologous DNA served as a recombinational anchor facilitating illegitimate recombination acting on the same molecule. Homologous stretches down to 183 nucleotides served as anchors. Transformation with heteroduplex DNA having different nucleotide sequence tags in the strands indicated that strands entered the cytoplasm 3' to 5' and that strands with either polarity were integrated by homologous recombination. The process led to the genomic integration of thousands of foreign nucleotides and often was accompanied by deletion of a roughly corresponding length of recipient DNA. Homology-facilitated illegitimate recombination would explain the introgression of DNA in prokaryotic genomes without the help of mobile genetic elements.     

Ability of RecA protein to promote a search for rare sequences in duplex DNA.

RecA nucleoprotein filaments found homologous targets even when the latter was mixed with 200,000 times as much heterologous duplex DNA. By contrast, mixing of the single-stranded probe with only 100 times as much heterologous single strands markedly reduced the rate of finding homologous duplex molecules. Titration of the reaction with different proportions of homologous single-stranded DNA distinguished a condition under which the search for homology itself was rate limiting from a condition under which some later step was limiting. Less than 1 min was required to scan 6.4 kilobase pairs of duplex DNA for homology to a RecA-coated single strand of the same size, but these experiments revealed that rapid searching by RecA nucleoprotein filaments was largely confined to neighboring duplex molecules. These observations provide guidelines for the use of RecA protein in locating rare sequences in complex mixtures of duplex DNA, and we describe a simple protocol by which rare sequences can be rapidly enriched at least a thousandfold.     

Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination.

Genomic double-strand breaks (DSBs) are key intermediates in recombination reactions of living organisms. We studied the repair of genomic DSBs by homologous sequences in plants. Tobacco plants containing a site for the highly specific restriction enzyme I-Sce I were cotransformed with Agrobacterium strains carrying sequences homologous to the transgene locus and, separately, containing the gene coding for the enzyme. We show that the induction of a DSB can increase the frequency of homologous recombination at a specific locus by up to two orders of magnitude. Analysis of the recombination products demonstrates that a DSB can be repaired via homologous recombination by at least two different but related pathways. In the major pathway, homologies on both sides of the DSB are used, analogous to the conservative DSB repair model originally proposed for meiotic recombination in yeast. Homologous recombination of the minor pathway is restricted to one side of the DSB as described by the nonconservative one-sided invasion model. The sequence of the recombination partners was absolutely conserved in two cases, whereas in a third case, a deletion of 14 bp had occurred, probably due to DNA polymerase slippage during the copy process. The induction of DSB breaks to enhance homologous recombination can be applied for a variety of approaches of plant genome manipulation.