Intermolecular homologous recombination in plants.

To study DNA topological requirements for homologous recombination in plants, we have constructed pairs of plasmids that contain nonoverlapping deletions in the neomycin phosphotransferase gene [APH(3')II], which, when intact, confers kanamycin resistance to plant cells. Protoplasts isolated from Nicotiana tabacum were cotransformed with complementary pairs of plasmids containing these truncated gene constructs. Homologous recombination or gene conversion within the homologous sequences (6 to 405 base pairs) of the protein-coding region of the truncated genes led to the restoration of the functional APH(3')II gene, rendering these cells resistant to kanamycin. Circular plasmid DNAs recombined very inefficiently, independent of the length of the homologous region. A double-strand break in one molecule only slightly increased the recombination frequency. The most favorable substrates for recombination were linear molecules. In this case, the recombination frequency was positively correlated with the length of the homologous regions. The recombination frequency of plasmids linearized at sites proximal to the deletion-homology junction was significantly higher than when linearization was distal to the homologous region. Vector homology within cotransformed plasmid sequences also increased the recombination frequency.     

The dual role of HOP2 in mammalian meiotic homologous recombination

Deletion of Hop2 in mice eliminates homologous chromosome synapsis and disrupts double-strand break (DSB) repair through homologous recombination. HOP2 in vitro shows two distinctive activities: when it is incorporated into a HOP2–MND1 complex it stimulates DMC1 and RAD51 recombination activities and the purified HOP2 alone is proficient in promoting strand invasion. We observed that a fraction of Mnd1^−/− spermatocytes, which express HOP2 but apparently have inactive DMC1 and RAD51 due to lack of the HOP2–MND1 complex, exhibits a high level of chromosome synapsis and that most DSBs in these spermatocytes are repaired. This suggests that DSB repair catalyzed solely by HOP2 supports homologous chromosome pairing and synapsis. In addition, we show that in vitro HOP2 promotes the co-aggregation of ssDNA with duplex DNA, binds to ssDNA leading to unstacking of the bases, and promotes the formation of a three-strand synaptic intermediate. However, HOP2 shows distinctive mechanistic signatures as a recombinase. Namely, HOP2-mediated strand exchange does not require ATP and, in contrast to DMC1, joint molecules formed by HOP2 are more sensitive to mismatches and are efficiently dissociated by RAD54. We propose that HOP2 may act as a recombinase with specific functions in meiosis.     

RecQ helicase enhances homologous recombination in plants

RecQ helicase is a key component in the RecF pathway of Escherichia coli for initiation of homologous recombination. Here, we demonstrate that transient expression of RecQ gene in rice embryogenic cell increases the homologous recombination efficiency as much as 4-fold. Further experiments reveal that this effect is influenced by the RecQ dosage. Stable expression of RecQ in rice dramatically increases the homologous recombination events 20- to 40-fold in leaf tissue from different transgenic lines. This is the first evidence indicating that overexpression of RecQ gene can stimulate homologous recombination in plants.     

Intrachromosomal homologous recombination in whole plants.

A system to assay intrachromosomal homologous recombination during the complete life-cycle of a whole higher eukaryote was set up. Arabidopsis thaliana plants were transformed with a recombination substrate carrying a non-selectable and quantitatively detectable marker gene. The recombination substrates contain two overlapping, non-functional deletion mutants of a chimeric beta-glucuronidase (uidA) gene. Upon recombination, as proven by Southern blot analysis, a functional gene is restored and its product can be detected by histochemical staining. Therefore, cells in which recombination events occurred, and their progeny, can be precisely localized in the whole plant. Recombination was observed in all plant organs examined, from the seed stage until the flowering stage of somatic plant development. Meristematic recombination events revealed cell lineage patterns. Overall recombination frequencies typically were in the range 10(-6)-10(-7) events/genome. Recombination frequencies were found to differ in different organs of particular transgenic lines.     

Gene replacement by homologous recombination in plants

After the elucidation of the sequence of the yeast genome a major effort was started to elucidate the biological function of all open reading frames of this organisms by targeted gene replacement via homologous recombination. The establishment of the complete sequence of the genome of Arabidopsis thaliana would principally allow a similar approach. However, over the past dozen years all attempts to establish an efficient gene targeting technique in flowering plants were in the end not successful. In contrast, in Physcomitrella patens an efficient gene targeting procedure has been set up, making the moss a valuable model system for plant molecular biologists. But also for flowering plants recently several new approaches – some of them based on the availability of the genomic sequence of Arabidopsis – were initiated that might finally result on the set up of a general applicable technique. Beside the production of hyper-recombinogenic plants either via expression or suppression of specific gene functions or via undirected mutagenesis, the application of chimeric oligonucleotides might result in major progress.     

Induction of intrachromosomal homologous recombination in whole plants

The influence of different factors on frequencies of intrachromosomal homologous recombination in whole Arabidopsis thaliana and tobacco plants was analyzed using a disrupted β-glucuronidase marker gene. Recombination frequencies were enhanced severalfold by DNA damaging agents like UV-light or MMS (methyl methanesulfonate). Applying 3-methoxybenzamide (3-MB), an inhibitor of poly(ADP)ribose polymerase (PARP), an enzyme that is postulated to be involved in DNA repair, enhanced homologous recombination frequencies strongly. These findings indicate that homologous recombination is involved in DNA repair and can (at least partially) compensate for other DNA repair pathways. Indications that recombination in plants can be induced by environmental stress factors that are not likely to be involved in DNA metabolism were also found; Arabidopsis plants growing in a medium containing 0.1 M NaCl exhibited elevated recombination frequencies. The possible general effects of ‘environmental’ challenges on genome flexibility are discussed.     

A tool for understanding homologous recombination in plants

Attempts for establishing an efficient gene targeting (GT) system in seed plants have hitherto not been successful. In contrast, GT based on homologous recombination is highly efficient in Physcomitrella, making this moss a novel tool in reverse genetics. However, why homologous and illegitimate recombination are differently regulated between Physcomitrella and seed plants is still enigmatic. Here we update the state of the art of GT in Physcomitrella and discuss approaches to unravel this enigma. Identification of molecular factors significantly enhancing GT and their subsequent transfer to crop plants will have a great impact on plant biotechnology by enabling precise genetic engineering. Physcomitrella appears to be the most useful model system in this context.     

The chromatin assembly factor subunit FASCIATA1 is involved in homologous recombination in plants

DNA replication in cycling eukaryotic cells necessitates the reestablishment of chromatin after nucleosome redistribution from the parental to the two daughter DNA strands. Chromatin assembly factor 1 (CAF-1), a heterotrimeric complex consisting of three subunits (p150/p60/p48), is one of the replication-coupled assembly factors involved in the reconstitution of S-phase chromatin. CAF-1 is required in vitro for nucleosome assembly onto newly replicated chromatin in human cells and Arabidopsis thaliana, and defects in yeast (Saccharomyces cerevisiae) affect DNA damage repair processes, predominantly those involved in genome stability. However, in vivo chromatin defects of caf-1 mutants in higher eukaryotes are poorly characterized. Here, we show that fasciata1-4 (fas1-4), a new allele of the Arabidopsis fas1 mutant defective in the p150 subunit of CAF-1, has a severe developmental phenotype, reduced heterochromatin content, and a more open conformation of euchromatin. Most importantly, homologous recombination (HR), a process involved in maintaining genome stability, is increased dramatically in fas1-4, as indicated by a 96-fold stimulation of intrachromosomal HR. Together with the open conformation of chromatin and the nearly normal expression levels of HR genes in the mutant, this result suggests that chromatin is a major factor restricting HR in plants.     

Role of the AtRad1p endonuclease in homologous recombination in plants

Using a specific recombination assay, we show in the plant Arabidopsis thaliana that AtRad1 protein plays a role in the removal of non-homologous tails in homologous recombination. Recombination in the presence of non-homologous overhangs is reduced 11-fold in the atrad1 mutant compared with the wild-type plants. AtRad1p is the A. thaliana homologue of the human Xpf and Saccharomyces cerevisiae Rad1 proteins. Rad1p is a subunit of the Rad1p/Rad10p structure-specific endonuclease that acts in nucleotide excision repair and inter-strand crosslink repair. This endonuclease also plays a role in mitotic recombination to remove non-homologous, 3′-ended overhangs from recombination intermediates. The Arabidopsis atrad1 mutant (uvh1), unlike rad1 mutants known from other eukaryotes, is hypersensitive to ionizing radiation. This last observation may indicate a more important role for the Rad1/Rad10 endonuclease in recombination in plants. This is the first direct demonstration of the involvement of AtRad1p in homologous recombination in plants.     

The nature of genetic recombination

The biological evolutionary axiom proposed earlier by the author states that in the absence of genetic recombination the evolution of organic forms would be impossible. In the present paper the literature data are considered, illustrating the role of genetic recombination in evolution. It is urged that a tendency towards an increasing complexity of biological organization results from periodical recombinational combining of the diverged genes as well as the whole genomes of different origin. The alternative mechanism implying the production of duplications from the identical gene copies or whole genomes is considered to be unlikely. According to the biological evolutionary axiom, the origin of life is connected with the appearance of a mode of reparation of crystalline type aggregates--the precursors of DNA by means of exchanges among their constituents. A hypothesis is proposed that in the process of recombination a certain distribution of the 6-amino bases (adenine, cytosine) along the DNA molecule is settled, with respect to the 6-carbonyl bases (guanine, thymine). It is proposed that the relative distribution of the bases mentioned influences electrostatic stability of the DNA molecule as a crystalline associate.     

The dual nature of neurotrophins

Neurotrophins are a small family of dimeric secretory proteins in vertebrate neurons with a broad spectrum of functions. They are generated as pro-proteins with a functionality that is distinct from the proteolytically processed form. The cellular responses of neurotrophins are mediated by three different types of receptor proteins, the receptor tyrosine kinases of the Trk family, the neurotrophin receptor p75(NTR), which is a member of the tumor necrosis factor receptor (TNFR) superfamily, and sortilin, previously characterized as neurotensin receptor. Recent studies have revealed an intriguing pattern: neurotrophins can elicit opposing signals utilising their variable configuration and different receptor types.     

A dual role for mycobacterial RecO in RecA-dependent homologous recombination and RecA-independent single-strand annealing

Mycobacteria have two genetically distinct pathways for the homology-directed repair of DNA double-strand breaks: homologous recombination (HR) and single-strand annealing (SSA). HR is abolished by deletion of RecA and reduced in the absence of the AdnAB helicase/nuclease. By contrast, SSA is RecA-independent and requires RecBCD. Here we examine the function of RecO in mycobacterial DNA recombination and repair. Loss of RecO elicits hypersensitivity to DNA damaging agents similar to that caused by deletion of RecA. We show that RecO participates in RecA-dependent HR in a pathway parallel to the AdnAB pathway. We also find that RecO plays a role in the RecA-independent SSA pathway. The mycobacterial RecO protein displays a zinc-dependent DNA binding activity in vitro and accelerates the annealing of SSB-coated single-stranded DNA. These findings establish a role for RecO in two pathways of mycobacterial DNA double-strand break repair and suggest an in vivo function for the DNA annealing activity of RecO proteins, thereby underscoring their similarity to eukaryal Rad52.     

Keeping homologous recombination in check

Pathway choice is a critical event in the repair of DNA double-strand breaks. In a recent paper published in Nature, Orthwein et al. define a mechanism by which homologous recombination is controlled in G1 cells to favor non-homologous end joining.Homologous recombination (HR) is an essential process that produces genetic variation during meiosis and protects the genome during mitotic cell division¹. Inherited mutations in various HR factors, including the BRCA1, BRCA2 and PALB2 tumor suppressors, predispose to the development of cancer. Although HR is generally beneficial for maintaining genome integrity, HR events between homologous chromosomes can also be deleterious and lead to loss of genetic information. HR is therefore suppressed during G1 phase and in non-dividing cells, yet, the exact mechanism behind this phenomenon has remained elusive. New work from the laboratory of Daniel Durocher describes a mechanism that is both necessary and sufficient for the suppression of HR in G1 cells².DNA double-strand breaks (DSBs) are one of the most dangerous types of DNA lesion and need to be eliminated to prevent the accumulation of mutations. DSB repair is carried out by two main pathways, HR and non-homologous end joining (NHEJ)¹. Whereas NHEJ is an error-prone process that simply fuses the two broken ends together, HR is essentially error-free as it uses the genetically identical sister chromatid as a template for repair. Due to the cell cycle-dependent availability of sister chromatids, HR is restricted to the S and G2 phases of the cell cycle.In the HR repair pathway, the DSB ends are first resected to produce extended single-stranded DNA (ssDNA) tails by the coordinated actions of a series of helicase and nuclease activities (e.g., MRN, CtIP and EXO1)¹. CtIP plays a particularly important role in regulating resection, which is mediated through its interaction with BRCA1³. In the following cascade of events, BRCA1 interacts directly with the BRCA2-PALB2 complex, which in turn is recruited to the ssDNA where it acts as a chaperone that stimulates the formation of RAD51 nucleoprotein filaments that drive homology-directed HR repair to restore the integrity of the DNA^4,5.Whereas most HR events take place between the newly replicated sister chromatids, recombination between homologous chromosomes can result in loss of heterozygosity, a potentially mutagenic event that can lead to the inactivation of tumor suppressors or activation of oncogenes. HR must therefore be tightly regulated and effectively suppressed in G1 phase, at the time when only homologous chromosomes are available for repair. At such times, NHEJ is the favored mechanism for DSB repair.A number of mechanisms regulate HR to a specific phase of the cell cycle. For example, CtIP is activated for interaction with BRCA1 by CDK-dependent phosphorylation, which occurs in the S and G2 phases of the cell cycle. Conversely, HR is suppressed in G1 phase by the pro-NHEJ factors 53BP1⁶, RIF1⁷ and REV7⁸, which impair the recruitment of BRCA1 and thereby inhibit DNA end resection. Consequently, disruption of 53BP1 leads to the recruitment of BRCA1 to DSBs in G1 phase. In the recent Nature paper from Durocher''s laboratory, Orthwein et al.² discovered that although BRCA1 is localized to DSBs during G1 phase in 53BP1-deficient cells, it fails to recruit the BRCA2-PALB2 complex, which is consistent with the lack of HR activity in these cells.Through immunoprecipitation experiments Orthwein et al. showed that while BRCA2 and PALB2 interact throughout the cell cycle, BRCA1 and PALB2 only interact efficiently in S phase, suggesting that there might be a mechanism that restricts their interaction to S and G2 phases, while also blocking it in G1 phase. The region of PALB2 that is responsible for its cell cycle-regulated interaction with BRCA1 was localized to its N-terminal domain, which corresponds to a known interaction site for KEAP1, a substrate adaptor for the CUL3-RING (CRL3) ubiquitin ligase. Remarkably, they found that deletion of the KEAP1 gene using CRISPR-Cas9 technology restored the BRCA1-PALB2 interaction in G1 cells, and led to the recruitment of BRCA2-PALB2 to sites of DNA damage in 53BP1-deficient G1 cells.Since KEAP1 is involved in protein ubiquitylation, Orthwein et al. hypothesized that ubiquitylation of PALB2 in the BRCA1-interacting region might block their interaction. Indeed, mutation of lysines in the interacting region of PALB2 restored its interaction with BRCA1 in G1 cells. Furthermore, pull-down experiments showed that ubiquitylation of PALB2 on Lysine-20 by KEAP1-CRL3 prevented its interaction with BRCA1. However, as neither the activity of the KEAP1-CRL3 ubiquitin ligase nor its interaction with BRCA1 is cell cycle regulated, Orthwein et al. reasoned that a deubiquitylation step could be the rate-limiting regulator of the BRCA1-PALB2 interaction. They highlighted the deubiquitylating enzyme USP11 as a potential candidate for this activity due to its interaction with BRCA1, BRCA2 and PALB2, and indeed found that USP11 disruption impaired the interaction between BRCA1 and PALB2. Moreover, they found that USP11 was unstable and interacted poorly with PALB2 in G1 cells, and that USP11 was rapidly lost by proteasomal degradation in G1 phase after DNA damage. By contrast, expression of USP11 in S-phase was high and insensitive to DNA damage. Taken together, these data led the authors to propose that the opposing activities of USP11 and KEAP1-CRL3 regulate cell cycle-dependent interactions between BRCA1 and PALB2 (Figure 1).Open in a separate windowFigure 1Schematic representation indicating how the opposing activities of USP11 and KEAP1-CRL3 regulate cell cycle-dependent interactions between BRCA1 and PALB2, and thereby mediate pathway choice in DSB repair.To extend these remarkable observations, Orthwein et al. disrupted this regulatory network to allow HR in G1 cells. They expected that depletion of KEAP1 in 53BP1-deficient cells might be sufficient for RAD51 foci formation following ionizing radiation (IR), but this was not the case because end resection remained a limiting factor. To counteract this, the authors expressed a constitutively active form of CtIP (T847E)⁹, which augmented resection and led to the efficient formation of IR-induced RAD51 foci in 53BP1- and KEAP1-deficient G1 cells. To address whether these RAD51 foci in G1 cells corresponded to productive HR events, they used a fluorescent-based gene-targeting assay. Whereas CtIP (T847E)expressed in 53BP1-deficient cells alone was insufficient to induce productive HR, depletion of KEAP1 or expression of a non-ubiquitylable version of PALB2 led to a robust increase in gene-targeting events. Collectively, this study therefore demonstrates that activation of DNA end resection, combined with the recruitment of BRCA2 to DSBs, are both necessary and sufficient to produce HR in G1 cells.Gene targeting has great potential for therapeutic purposes, but the fact that most cells in the body are non-dividing has so far limited its use¹⁰. We suspect that the new knowledge highlighted in this work will further improve gene-targeting therapies to help fight human diseases.     

Extensive homologous recombination between introduced and native regulatory plastid DNA elements in transplastomic plants

Homologous recombination within plastids directs plastid genome transformation for foreign gene expression and study of plastid gene function. Though transgenes are generally efficiently targeted to their desired insertion site, unintended homologous recombination events have been observed during plastid transformation. To understand the nature and abundance of these recombination events, we analyzed transplastomic tobacco lines derived from three different plastid transformation vectors utilizing two different loci for foreign gene insertion. Two unintended recombinant plastid DNA species were formed from each regulatory plastid DNA element included in the transformation vector. Some of these recombinant DNA species accumulated to as much as 10–60% of the amount of the desired integrated transgenic sequence in T0 plants. Some of the recombinant DNA species undergo further, “secondary” recombination events, resulting in an even greater number of recombinant plastid DNA species. The abundance of novel recombinant DNA species was higher in T0 plants than in T1 progeny, indicating that the ancillary recombination events described here may have the greatest impact during selection and regeneration of transformants. A line of transplastomic tobacco was identified containing an antibiotic resistance gene unlinked from the intended transgene insertion as a result of an unintended recombination event, indicating that the homologous recombination events described here may hinder efficient recovery of plastid transformants containing the desired transgene. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Increase of homologous recombination frequency in vascular tissue of Arabidopsis plants exposed to salt stress

Here we analyzed the influence of salt stress on plant genome stability. Homologous recombination events were detected in transgenic Arabidopsis plants that carried in their genome a beta-glucuronidase recombination marker. Recombination events were scored as blue sectors using a stereo microscope. Exposure to 50 mM salt resulted in a 3.0-fold increase in recombination frequency. To analyze the organ and tissue specificity of recombination events, we examined cross-sections of leaves, stems and roots. We found that nearly 30% of recombination events in plants grown under normal conditions and nearly 50% of events in plants grown on salt were undetected by the conventional method. Most of the recombination events represented a cluster/group of cells (12 on average), although events with single cells were also detected. Recombination events were very frequent in leaf mesophyll cells. On average, individual recombination events located on leaves contained more cells than events located on roots or stems. Analysis of recombination events in cross-sectioned tissue of salt-treated plants revealed a shift in the distribution of recombination events towards the vascular tissue. We discuss the significance of the finding for plant stress physiology.     

The frequency of homologous recombination in human ALT cells

Instead of telomerase, some immortal cells use the alternative lengthening of telomeres pathway (ALT) to maintain their telomeres. There is good evidence that homologous recombination contributes to the ALT mechanism. Using an inducible GFP reporter system to measure the frequency of homologous recombination, we asked whether or not ALT cells exhibited a general change of the recombination machinery. Our results show that the frequency of homologous recombination for non-telomeric sequences in ALT cells is identical to that in telomerase positive cells, irrespective of whether the reporter was present at an intra-chromosomal location or next to a telomeric sequence. We conclude that the underlying recombination defect in ALT cells is restricted to telomeric sequences.     

Ecological genomics in Xanthomonas: the nature of genetic adaptation with homologous recombination and host shifts

Background

Comparative genomics provides insights into the diversification of bacterial species. Bacterial speciation usually takes place with lasting homologous recombination, which not only acts as a cohering force between diverging lineages but brings advantageous alleles favored by natural selection, and results in ecologically distinct species, e.g., frequent host shift in Xanthomonas pathogenic to various plants.

Results

Using whole-genome sequences, we examined the genetic divergence in Xanthomonas campestris that infected Brassicaceae, and X. citri, pathogenic to a wider host range. Genetic differentiation between two incipient races of X. citri pv. mangiferaeindicae was attributable to a DNA fragment introduced by phages. In contrast to most portions of the genome that had nearly equivalent levels of genetic divergence between subspecies as a result of the accumulation of point mutations, 10% of the core genome involving with homologous recombination contributed to the diversification in Xanthomonas, as revealed by the correlation between homologous recombination and genomic divergence. Interestingly, 179 genes were under positive selection; 98 (54.7%) of these genes were involved in homologous recombination, indicating that foreign genetic fragments may have caused the adaptive diversification, especially in lineages with nutritional transitions. Homologous recombination may have provided genetic materials for the natural selection, and host shifts likely triggered ecological adaptation in Xanthomonas. To a certain extent, we observed positive selection nevertheless contributed to ecological divergence beyond host shifting.

Conclusion

Altogether, mediated with lasting gene flow, species formation in Xanthomonas was likely governed by natural selection that played a key role in helping the deviating populations to explore novel niches (hosts) or respond to environmental cues, subsequently triggering species diversification.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1369-8) contains supplementary material, which is available to authorized users.     

The recX gene potentiates homologous recombination in Neisseria gonorrhoeae

In the pathogen Neisseria gonorrhoeae (Gc), the RecA protein is necessary for DNA repair, DNA transformation and pilus antigenic variation. Many bacteria contain a gene, recX, which has been suggested to downregulate recA through an unknown mechanism. To investigate the possible role of recX in Gc, we cloned and insertionally inactivated the recX gene. The recX loss-of-function mutant showed decreases in pilus phase variation, DNA transformation and DNA repair ability compared with wild type. We were able to complement all these deficiencies by supplying a functional copy of recX elsewhere in the chromosome. The recX mutant still showed increases in pilus phase variation under conditions of iron starvation, and the recX mutant showed levels of RecA protein equivalent to wild type. Although the precise role of recX in recombination remains unclear, RecX aids all RecA-related processes in Gc, and this is the first demonstration of a role for recX in homologous recombination in any organism.