A transient assay for recombination demonstrates that Arabidopsis SNM1 and XRCC3 enhance non-homologous recombination

Replacement of endogenous genes by homologous recombination is rare in plants; the majority of genetic modifications are the result of transforming DNA molecules undergoing random genomic insertion by way of non-homologous recombination. Factors that affect chromatin remodeling and DNA repair are thought to have the potential to enhance the frequency of homologous recombination in plants. Conventional tools to study the frequencies of genetic recombination often rely on stable transformation-based approaches, with these systems being rarely capable of high-throughput or combinatorial analysis. We developed a series of vectors that use chemiluminescent (LUC and REN) reporter genes to assay the relative frequency of homologous and non-homologous recombination in plants. These transient assay vectors were used to screen 14 candidate genes for their effects on recombination frequencies in Nicotiana benthamiana plants. Over-expression of Arabidopsis genes with sequence similarity to SNM1 from yeast and XRCC3 from humans enhanced the frequency of non-homologous recombination when assayed using two different donor vectors. Transient N. benthamiana leaf systems were also used in an alternative assay for preliminary measurements of homologous recombination frequencies, which were found to be enhanced by over-expression of RAD52, MIM and RAD51 from yeast, as well as CHR24 from Arabidopsis. The findings for the assays described here are in line with previous studies that analyzed recombination frequencies using stable transformation. The assays we report have revealed functions in non-homologous recombination for the Arabidopsis SNM1 and XRCC3 genes, so the suppression of these genes' expression offers a potential means to enhance the gene targeting frequency in plants. Furthermore, our findings also indicate that plant gene targeting frequencies could be enhanced by over-expression of RAD52, MIM, CHR24, and RAD51 genes.     

Both CRISPR/Cas‐based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana

Engineered nucleases can be used to induce site‐specific double‐strand breaks (DSBs) in plant genomes. Thus, homologous recombination (HR) can be enhanced and targeted mutagenesis can be achieved by error‐prone non‐homologous end‐joining (NHEJ). Recently, the bacterial CRISPR/Cas9 system was used for DSB induction in plants to promote HR and NHEJ. Cas9 can also be engineered to work as a nickase inducing single‐strand breaks (SSBs). Here we show that only the nuclease but not the nickase is an efficient tool for NHEJ‐mediated mutagenesis in plants. We demonstrate the stable inheritance of nuclease‐induced targeted mutagenesis events in the ADH1 and TT4 genes of Arabidopsis thaliana at frequencies from 2.5 up to 70.0%. Deep sequencing analysis revealed NHEJ‐mediated DSB repair in about a third of all reads in T1 plants. In contrast, applying the nickase resulted in the reduction of mutation frequency by at least 740‐fold. Nevertheless, the nickase is able to induce HR at similar efficiencies as the nuclease or the homing endonuclease I–SceI. Two different types of somatic HR mechanisms, recombination between tandemly arranged direct repeats as well as gene conversion using the information on an inverted repeat could be enhanced by the nickase to a similar extent as by DSB‐inducing enzymes. Thus, the Cas9 nickase has the potential to become an important tool for genome engineering in plants. It should not only be applicable for HR‐mediated gene targeting systems but also by the combined action of two nickases as DSB‐inducing agents excluding off‐target effects in homologous genomic regions.     

Methylation Status of Arabidopsis DNA Repair Gene Promoters During Agrobacterium Infection Reveals Epigenetic Changes in Three Generations

Agrobacterium tumefaciens is a unique pathogen with the ability to transfer a portion of its DNA, the T-DNA, to other organisms. The role of DNA repair genes in Agrobacterium transformation remains controversial. In order to understand if the host DNA repair response and dynamics was specific to bacterial factors such as Vir proteins, T-DNA, and oncogenes, we profiled the expression and promoter methylation of various DNA repair genes. These genes belonged to nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), homologous recombination (HR), and non-homologous end joining (NHEJ) pathways. We infected Arabidopsis plants with different Agrobacterium strains that lacked one or more of the above components so that the influence of the respective factors could be analysed. Our results revealed that the expression and promoter methylation of most DNA repair genes was affected by Agrobacterium, and it was specific to Vir proteins, T-DNA, oncogenes, or the mere presence of bacteria. In order to determine if Agrobacterium induced any transgenerational epigenetic effect on the DNA repair gene promoters, we studied the promoter methylation in two subsequent generations of the infected plants. Promoters of at least three genes, CEN2, RAD51, and LIG4 exhibited transgenerational memory in response to different bacterial factors. We believe that this is the first report of Agrobacterium-induced transgenerational epigenetic memory of DNA repair genes in plants. In addition, we show that Agrobacterium induces short-lived DNA strand breaks in Arabidopsis cells, irrespective of the presence or absence of virulence genes and T-DNA.

     

Genistein-induced DNA damage is repaired by nonhomologous end joining and homologous recombination in TK6 cells

Genistein (GES), a phytoestrogen, has potential chemopreventive and chemotherapeutic effects on cancer. The anticancer mechanism of GES may be related with topoisomerase II associated DNA double-strand breaks (DSBs). However, the precise molecular mechanism remains elusive. Here, we performed genetic analyses using human lymphoblastoid TK6 cell lines to investigate whether non-homologous DNA end joining (NHEJ) and homologous recombination (HR), the two major repair pathways of DSBs, were involved in repairing GES-induced DNA damage. Our results showed that GES induced DSBs in TK6 cells. Cells lacking Ligase4, an NHEJ enzyme, are hypersensitive to GES. Furthermore, the sensitivity of Ligase4^−/− cells was associated with enhanced DNA damage when comparing the accumulation of γ-H2AX foci and number of chromosomal aberrations (CAs) with WT cells. In addition, cells lacking Rad54, a HR enzyme, also presented hypersensitivity and increased DNA damages in response to GES. Meanwhile, Treatment of GES-lacking enhanced the accumulation of Rad51, an HR factor, in TK6 cells, especially in Ligase4^−/−. These results provided direct evidence that GES induced DSBs in TK6 cells and clarified that both NHEJ and HR were involved in the repair of GES-induced DNA damage, suggesting that GES in combination with inhibition of NHEJ or HR would provide a potential anticancer strategy.     

Elevated levels of intrachromosomal homologous recombination in Arabidopsis overexpressing the MIM gene

The Arabidopsis MIM gene encodes a protein belonging to the SMC family (structure maintenance of chromosomes) which is required for intrachromosomal homologous recombination (ICR). Both ICR and MIM gene expression are enhanced by DNA-damaging treatments, suggesting that MIM is a factor limiting DNA repair by homologous recombination (HR) under genotoxic stress. We tested this hypothesis by measuring the levels of recombination in the mim mutant under genotoxic stress, using methyl methanesulfonate. Although the mutant clearly showed diminished basal and induced levels of ICR, enhancement of ICR by DNA-damaging treatments was similar to that observed in the wild type. This suggests that the MIM gene product is required for DNA repair by HR, but is not critical for HR induction. To determine whether enhanced availability of MIM would increase basal HR levels in Arabidopsis, we examined ICR frequencies in transgenic Arabidopsis strains overexpressing the MIM gene after ectopic insertion of additional MIM copies. Two independent lines showed a twofold increase in ICR frequency relative to the wild type. Thus MIM is required for efficient ICR in plants, and its manipulation can be used to change homologous recombination frequencies. Since MIM is one of the components responsible for chromatin dynamics, our results suggest that the chromatin environment determines the frequency of homologous recombination.     

Bacillus subtilis SbcC protein plays an important role in DNA inter-strand cross-link repair

Background  

Several distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs) can be repaired through direct joining of broken ends (non homologous end joining, NHEJ) or through recombination with the non broken sister chromosome (homologous recombination, HR). Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells.     

In vivo biochemistry: Physical monitoring of recombination induced by site-specific endonucleases

The recombinational repair of chromosomal double-strand breaks (DSBs) is of critical importance to all organisms, who devote considerable genetic resources to ensuring such repair is accomplished. In Saccharomyces cerevisiae, DSB-mediated recombination can be initiated synchronously by the conditional expression of two site-specific endonucleases, HO or I-Scel. DNA undergoing recombination can then be extracted at intervals and analyzed. Recombination initiated by meiotic-specific DSBs can be followed in a similar fashion. This type of ‘in vivo biochemistry’ has been used to describe several discrete steps in two different homologous recombination pathways: gene conversion and single-strand annealing. The roles of specific proteins during recombination can be established by examining DNA in strains deleted for the corresponding gene. These same approaches are now becoming available for the study of recombination in both higher plants and animals. Physical monitoring can also be used to analyze nonhomologous recombination events, whose mechanisms appear to be conserved from yeast to mammals.     

MHF1 plays Fanconi anaemia complementation group M protein (FANCM)‐dependent and FANCM‐independent roles in DNA repair and homologous recombination in plants

Fanconi anaemia complementation group M protein (FANCM), a component of the human Fanconi anemia pathway, acts as DNA translocase that is essential during the repair of DNA interstrand cross‐links. The DNA‐damage‐binding function of FANCM is strongly enhanced by the histone fold‐containing FANCM‐associated protein MHF1. We identified a single homologue of MHF1 in the genome of Arabidopsis thaliana. Similar to the loss of AtFANCM, the loss of AtMHF1 leads to several meiotic defects, such as chromosome bridges between bivalents and an unequal distribution of chromosomes. Moreover, MHF1, together with FANCM, is involved in interstrand cross‐link repair in plants. This phenotype is detectable only in double mutants of the RecQ helicase and BLM homologue RECQ4A, which appears to function in a parallel pathway to the FANCM/MHF1 complex. However, in somatic cells, FANCM has an MHF1‐independent function in replicative repair in a parallel pathway to the endonuclease MUS81. Furthermore, MHF1 is required for efficient somatic homologous recombination (HR) – a role antagonistic to FANCM. FANCM and RECQ4A define two parallel pathways of HR suppression in Arabidopsis. Hyperrecombination in the fancm but not the recq4A mutant can be abolished by MHF1 mutations. This finding indicates that MHF1 and FANCM act at different steps of a single, common, HR pathway.     

Different functional roles of RTR complex factors in DNA repair and meiosis in Arabidopsis and tomato

The RTR (R ecQ/Top 3/Rmi 1) complex has been elucidated as essential for ensuring genome stability in eukaryotes. Fundamental for the dissolution of Holliday junction (HJ)-like recombination intermediates, the factors have been shown to play further, partly distinct roles in DNA repair and homologous recombination. Across all kingdoms, disruption of this complex results in characteristic phenotypes including hyper-recombination and sensitivity to genotoxins. The type IA topoisomerase TOP3α has been shown as essential for viability in various animals. In contrast, in the model plant species Arabidopsis, the top3α mutant is viable. rmi1 mutants are deficient in the repair of DNA damage. Moreover, as opposed to other eukaryotes, TOP3α and RMI1 were found to be indispensable for proper meiotic progression, with mutants showing severe meiotic defects and sterility. We now established mutants of both TOP3α and RMI1 in tomato using CRISPR/Cas technology. Surprisingly, we found phenotypes that differed dramatically from those of Arabidopsis: the top3α mutants proved to be embryo-lethal, implying an essential role of the topoisomerase in tomato. In contrast, no defect in somatic DNA repair or meiosis was detectable for rmi1 mutants in tomato. This points to a differentiation of function of RTR complex partners between plant species. Our results indicate that there are relevant differences in the roles of basic factors involved in DNA repair and meiosis within dicotyledons, and thus should be taken as a note of caution when generalizing knowledge regarding basic biological processes obtained in the model plant Arabidopsis for the entire plant kingdom.     

Reactive oxygen species stimulate homologous recombination in plants

Coping with the continuous production of free radicals is a daily routine of the cell. Despite their toxicity, the reactive oxygen species (ROS) are involved in dual physiological action – signal transduction and immune response. We analysed the influence of oxidative stress‐generating compounds, rose Bengal (RB), paraquat (PQ) and amino‐triazole (ATZ) on the genome stability of Arabidopsis using transgenic recombination‐monitoring plants. Homologous recombination frequencies in plants were increased upon the treatment with RB and PQ but not ATZ. Application of the N‐acetyl‐L ‐cysteine (NAC), radicals scavenging compound, decreased the DNA damage caused by RB. Interestingly, the incubation of plants with very low concentration of RB (less than 0.2 µM ) led to the subsequent increase in plant tolerance to methyl methane sulfonate (MMS): stronger plants with a lower increase of homologous recombination frequency. In contrast, the incubation of plants with 0.5 µM of RB resulted in the potentiation of the MMS effect: the weaker plants with higher frequency of recombination. The data of the present study suggest the existence of a dual concentration‐dependent role of ROS in plants.     

High‐efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9

The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence‐specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T‐DNA, biolistics or by stably integrating nuclease‐encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10‐fold enhancements in GT frequencies have been achieved using DNA virus‐based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon‐based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110‐fold increase in expression of a reporter gene relative to non‐replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12‐fold greater than non‐viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene‐targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV‐based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.     

Requirement for the SRS2 DNA helicase gene in non-homologous end joining in yeast

Mitotic cells experience double-strand breaks (DSBs) from both exogenous and endogenous sources. Since unrepaired DSBs can result in genome rearrangements or cell death, cells mobilize multiple pathways to repair the DNA damage. In the yeast Saccharomyces cerevisiae, mitotic cells preferentially use a homologous recombination repair pathway. However, when no significant homology to the DSB ends is available, cells utilize a repair process called non-homologous end joining (NHEJ), which can join ends with no homology through resection to uncover microhomologies of a few nucleotides. Although components of the homologous recombination repair system are also involved in NHEJ, the rejoining does not involve all of the homologous recombination repair genes. The SRS2 DNA helicase has been shown to be required for DSB repair when the homologous single-stranded regions are short. Here it is shown that SRS2 is also required for NHEJ, regardless of the cell mating type. Efficient NHEJ of sticky ends requires the Ku70 and Ku80 proteins and the silencing genes SIR2, SIR3 and SIR4. However, NHEJ of blunt ends, while very inefficient, is not further reduced by mutations in YKU70, SIR2, SIR3, SIR4 or SRS2, suggesting that this rejoining process occurs by a different mechanism.     

Different functions for the domains of the Arabidopsis thaliana RMI1 protein in DNA cross-link repair,somatic and meiotic recombination

Recombination intermediates, such as double Holliday junctions, can be resolved by nucleases or dissolved by the combined action of a DNA helicase and a topoisomerase. In eukaryotes, dissolution is mediated by the RTR complex consisting of a RecQ helicase, a type IA topoisomerase and the structural protein RecQ-mediated genome instability 1 (RMI1). Throughout eukaryotes, the RTR complex is involved in DNA repair and in the suppression of homologous recombination (HR) in somatic cells. Surprisingly, Arabidopsis thaliana mutants of topoisomerase 3α and RMI1 are also sterile due to extensive chromosome breakage in meiosis I, indicating that both proteins are essential for meiotic recombination in plants. AtRMI1 harbours an N-terminal DUF1767 domain and two oligosaccharide binding (OB)-fold domains. To define specific roles for these individual domains, we performed complementation experiments on Atrmi1 mutants with an AtRMI1 full-length open reading frame (ORF) or deletion constructs lacking specific domains. We show that the DUF1767 domain and the OB-fold domain 1 are both essential for the function of AtRMI1 in DNA cross-link repair as well as meiotic recombination, but partially dispensable for somatic HR suppression. The OB-fold domain 2 is not necessary for either somatic or meiotic HR, but it seems to have a minor function in DNA cross-link repair.     

Recombination in the Plant Genome and its Application in Biotechnology

Genetic variation within and between species is based on recombination of DNA molecules. Recombination also plays a very important role in the repair of damaged DNA. Clarity about the mechanism by which recombination occurs is of profound interest not only to understand how this process assures the maintenance of genome integrity and at the same time is the driving force of evolution, but also for its application in biotechnology. The isolation of genes involved in recombination and the elucidation of the role of many of the corresponding gene products in Escherichia coli and Saccharomyces cerevisiae has formed the basis for comparative analysis in other, more complex eukaryotic systems. The identification of homologous genes from different organisms, including plants, suggests a conservation of the general mechanisms of recombination. Transgenes introduced in an organism may be incorporated in the genome by either homologous or nonhomologous recombination (end joining). The preferred pathway differs strongly between organisms. In plants there is a preference for random integration of the introduced DNA by nonhomologous recombination, which might lead to the accidental inactivation of important genes and to variable and unpredictable expression of the transgene itself. Therefore, there is an urgent need for the development and improvement of techniques for the directed integration of transgenes at specific locations in the genome. The integration of transgenes by homologous recombination would allow specific modification or disruption of endogenous genes, providing a tool for more detailed analysis of gene function. In combination with the recent introduction of site-specific recombination systems from E. coli or yeast into plants, this may lead to the development of versatile systems for modification of the plant genome.     

Hypermutation in stationary-phaseE. coli: tales from thelac operon

Escherichia coli cells are capable of complex regulatory responses to environmental conditions and stresses. In some circumstances, the response includes an increase in the mutation rate, effectively mutagenizing the genome. The classic example is the SOS response to DNA damage. Recent work indicates that other environmental stresses can also result in mutation of the genome. Modulation of mutation rate may be a more prevalent stress response than previously thought. In this review we focus on genome-wide mutation inE. coli cells subjected to a nonlethal genetic selection for reversion of alac frameshift allele. Reversion of thelac frameshift allele occurs via a novel mechanism that requires homologous recombination functions, and is enhanced by transiently diminished postsynthesis mismatch repair. A model for recombination-dependent stationary-phase mutation will be presented and its relevance for genome-wide mutation discussed.     

Specific targeted gene repair using single-stranded DNA oligonucleotides at an endogenous locus in mammalian cells uses homologous recombination

The feasibility of introducing point mutations in vivo using single-stranded DNA oligonucleotides (ssON) has been demonstrated but the efficiency and mechanism remain elusive and potential side effects have not been fully evaluated. Understanding the mechanism behind this potential therapy may help its development. Here, we demonstrate the specific repair of an endogenous non-functional hprt gene by a ssON in mammalian cells, and show that the frequency of such an event is enhanced when cells are in S-phase of the cell cycle. A potential barrier in using ssONs as gene therapy could be non-targeted mutations or gene rearrangements triggered by the ssON. Both the non-specific mutation frequencies and the frequency of gene rearrangements were largely unaffected by ssONs. Furthermore, we find that the introduction of a mutation causing the loss of a functional endogenous hprt gene by a ssON occurred at a similarly low but statistically significant frequency in wild type cells and in cells deficient in single strand break repair, nucleotide excision repair and mismatch repair. However, this mutation was not induced in XRCC3 mutant cells deficient in homologous recombination. Thus, our data suggest ssON-mediated targeted gene repair is more efficient in S-phase and involves homologous recombination.     

The Arabidopsis thaliana Homolog of the Helicase RTEL1 Plays Multiple Roles in Preserving Genome Stability

In humans, mutations in the DNA helicase Regulator of Telomere Elongation Helicase1 (RTEL1) lead to Hoyeraal-Hreidarsson syndrome, a severe, multisystem disorder. Here, we demonstrate that the RTEL1 homolog in Arabidopsis thaliana plays multiple roles in preserving genome stability. RTEL1 suppresses homologous recombination in a pathway parallel to that of the DNA translocase FANCM. Cytological analyses of root meristems indicate that RTEL1 is involved in processing DNA replication intermediates independently from FANCM and the nuclease MUS81. Moreover, RTEL1 is involved in interstrand and intrastrand DNA cross-link repair independently from FANCM and (in intrastrand cross-link repair) parallel to MUS81. RTEL1 contributes to telomere homeostasis; the concurrent loss of RTEL1 and the telomerase TERT leads to rapid, severe telomere shortening, which occurs much more rapidly than it does in the single-mutant line tert, resulting in developmental arrest after four generations. The double mutant rtel1-1 recq4A-4 exhibits massive growth defects, indicating that this RecQ family helicase, which is also involved in the suppression of homologous recombination and the repair of DNA lesions, can partially replace RTEL1 in the processing of DNA intermediates. The requirement for RTEL1 in multiple pathways to preserve genome stability in plants can be explained by its putative role in the destabilization of DNA loop structures, such as D-loops and T-loops.     

A RAD52‐like single‐stranded DNA binding protein affects mitochondrial DNA repair by recombination

The plant mitochondrial DNA‐binding protein ODB1 was identified from a mitochondrial extract after DNA‐affinity purification. ODB1 (organellar DNA‐binding protein 1) co‐purified with WHY2, a mitochondrial member of the WHIRLY family of plant‐specific proteins involved in the repair of organellar DNA. The Arabidopsis thaliana ODB1 gene is identical to RAD52‐1, which encodes a protein functioning in homologous recombination in the nucleus but additionally localizing to mitochondria. We confirmed the mitochondrial localization of ODB1 by in vitro and in vivo import assays, as well as by immunodetection on Arabidopsis subcellular fractions. In mitochondria, WHY2 and ODB1 were found in large nucleo‐protein complexes. Both proteins co‐immunoprecipitated in a DNA‐dependent manner. In vitro assays confirmed DNA binding by ODB1 and showed that the protein has higher affinity for single‐stranded than for double‐stranded DNA. ODB1 showed no sequence specificity in vitro. In vivo, DNA co‐immunoprecipitation indicated that ODB1 binds sequences throughout the mitochondrial genome. ODB1 promoted annealing of complementary DNA sequences, suggesting a RAD52‐like function as a recombination mediator. Arabidopsis odb1 mutants were morphologically indistinguishable from the wild‐type, but following DNA damage by genotoxic stress, they showed reduced mitochondrial homologous recombination activity. Under the same conditions, the odb1 mutants showed an increase in illegitimate repair bypasses generated by microhomology‐mediated recombination. These observations identify ODB1 as a further component of homologous recombination‐dependent DNA repair in plant mitochondria.