WO2013072914A2 - Plant rad52 and uses thereof - Google Patents

Abstract

The present invention relates to compositions and methods useful in genetic manipulation of plants. Particularly, the present invention discloses the presence of RAD52 analogues in plants and provides genetically modified plants having altered homologous recombination and DNA repair processes.

Description

PLANT RAD52 AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to compositions and methods useful in genetic manipulation of plants, particularly to polynucleotides encoding plant RAD52 analogues and use thereof for modulating recombination and DNA repair processes in plants.

BACKGROUND OF THE INVENTION

DNA in plants is prone to damages from environmental stresses such as ultraviolet (UV) radiation and chemical substances, and to errors that occur during DNA replication. These processes may cause cell death and undesirable effects on the plant growth and yield. Several DNA repair pathways have evolved in plants and include photoreactivation, nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), trans lesion synthesis (TLS), interstrand crosslink repair (ICL repair) and double-strand break repair (DSB repair). Double-strand breaks (DSBs) in the DNA are repaired by two essential mechanisms: homologous recombination (HR) and nonhomologous end-joining (NHEJ). In the homologous recombination process, the break is repaired through the recombination of homologous DNA regions, i.e. an identical or very similar DNA sequence is used as a template. The homologous recombination process is further divided to the DNA double-strand break repair (DSBR) model which mostly occurs in meiotic cells and to the synthesis dependent strand annealing (SDSA) model, which mostly occurs in somatic cells. In the nonhomologous end-joining process, the ends of the broken DNA strands are joined directly, mostly independent of the sequence.

Homologous recombination is a well-established tool for gene targeting comprising the introduction of an exogenous homologous sequence (i.e. targeting vector) and its recognition by the cellular machinery, resulting in the recombination between the endogenous and exogenous homologous sequences. The end result is a targeting vector inserted into a specific chromosome while the rest of the genome is not altered. Gene targeting by homologous recombination is typically used to disrupt the expression of a particular gene, such as in the knockout (KO) process or by introducing subtle mutations into a target gene or replace it with a new gene in a process known as gene knockin (KI). Gene targeting by homologous recombination is considered a safe and efficient biological approach for gene replacement especially when compared to viral mediated approaches where random integration of the targeting vector may occur, resulting in activation or deactivation of endogenous genes that may lead to undesired effects.

RADiation sensitive52 (RAD52) protein is a member of a group of proteins involved in the repair of DNA DSBs and is also involved in the repair of other types of DNA lesions, such as stalled replication forks. RAD52 homologs have been identified in eukaryotic organisms including yeast and humans. However, genes or encoded proteins have not been identified to date in plants and invertebrates. The presence of two plant BRCA2 homologs has been proposed to compensate for the apparent absence of RAD52 homologs in plants. When present, the RAD52 protein has been shown to have a pivotal role in DNA DSB repair and in homologous recombination, by forming a heptameric ring, catalyzing DNA annealing and mediating the RAD51 -catalysed strand invasion.

Mechanistically, RAD52 is recruited to the Replication Protein A (RPA)-single stranded DNA nucleoprotein complex that is formed upon DSB induction and exonucleolytic ends resection, to protect the single strand (SS) DNA from nucleo lytic degradation and from the configuration of secondary structures. It then mediates the replacement of RPA by the RAD51 protein, which in turn catalyses strand invasion and D-loop formation. Eventually, RAD52 may help capture the second DNA end and promote its annealing to the D-loop, thus leading to the formation of Holliday junctions (Mortensen U. H. et al. 2009. Curr Biol 19, R676-677). RAD52 has several domains, each with a distinct molecular function. The N-terminal of RAD52 protein, which is considered as the most conserved domain across eukaryotic species, binds to RAD59 and has two self-association domains, allowing it to multimerize into a heptameric ring. The central region interacts with the replication protein A (RPA) and the third C-terminal region, which is considered to be the least conserved domain across species, binds RAD51 and catalyzes DNA annealing. U.S. Patent Application Publication No. 2003/0121074 discloses isolated nucleic acid molecules encoding plant XRCC3 proteins that are involved in homologous recombination and DNA-repair processes in plants. The patent application further discloses a method for altering recombination frequency in a plant comprising introducing into a plant the isolated nucleic acid molecules. U.S. Patent No. 6,906,243 discloses an isolated nucleic acid molecule comprising

MSH2 nucleotide sequences which encode the MSH2 protein. This protein has been identified in plants and has been shown to alter mismatch repair, mutation rates and recombination frequencies in both eukaryotic and prokaryotic organisms. The Patent also discloses a method for altering DNA repair processes in a plant comprising introducing into a plant the isolated MSH2 nucleotide sequence.

Additional reports of the identification and use of nucleic acid sequences encoding proteins involved in DNA repair and recombination machinery in plants have been disclosed, for example U.S. Patent No. 6,541,684 which provides nucleotide sequences encoding two active RAD51 recombinases in maize plants. Methods useful in increasing the efficiency of homologous recombination have also been disclosed (e.g. U.S. Patent No. 7,892,823) as well as methods for stimulating recombination in somatic cells, for example in U.S. Patent Nos. 5,945,339 and 5,780,296.

U.S. Patent Application Publication No. 2004/0111764 discloses a method for elevating the frequency of meiotic recombination in plants comprising expressing a polynucleotide encoding a recombinational DNA repair polypeptide capable of stimulating meiotic recombination, in particular the RAD51 protein.

A paper of the inventors of the present invention, published after the priority date of the present invention, describes the identification and characterization of plant RAD52 homologs (Samach A et al. 2011. The Plant Cell 23, 4266-4279).

There is an unmet need to identify and characterize nucleic acid molecules and proteins involved in DNA repair and recombination processes in plants. In particular, identification of novel proteins involved in homologous recombination would be highly advantageous to modulate the frequency of endogenous homologous recombination and increase the efficiency of gene targeting, thereby providing improved technologies for use in plants, particularly for plant breeding.

SUMMARY OF THE INVENTION The present invention discloses for the first time that RAD52 analogues are present in plants and affect DNA repair as well as homologous recombination processes. The present invention thus provides isolated polynucleotide sequences encoding two putative RAD52 protein analogues and methods for altering the frequency of homologous recombination and the efficiency of DNA repair processes in plants. The present invention further provides transgenic plants comprising the polynucleotides of the present invention, including male sterile plants.

The present invention is based in part on exceptional evidence including sequence, structure and functional data teaching the presence and function of the plant RAD52 analogue. Specifically, the present invention is based on the identification of two RAD52 analogue genes in Arabidopsis, designated AtRAD52-l and A tRAD52-2. The AtRAD52-l gene has three splice variants that encode two different active proteins (AtRAD52-lA and AtRAD52-lB). The AtRAD52-2 gene has two splice variants encoding for AtRAD52-2A and AtRAD52-2B. The present invention is further partially based on structural similarity between the plant RAD52 analogue and the human RAD52 proteins. In addition, the present invention is based on functional evidence that plant RAD52 analogues are involved in cellular functions including DNA repair and homologous recombination.

According to one aspect, the present invention provides a genetically modified plant having altered expression of at least one plant RAD52 analogue, wherein the plant RAD52 analogue has an amino acid sequence at least 60% homologous to the amino acid sequence set forth in any one of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or an active fragment thereof. Alteration of the plant RAD52 expression results in at least one of reduced recombination frequency; increased recombination frequency; reduced efficacy of DNA repair or enhanced efficacy of DNA repair compared to a corresponding unmodified plant.

According to certain embodiments, the genetically modified plant is a transgenic plant comprising at least one cell comprising a transcribable exogenous polynucleotide encoding the at least one plant RAD52 analogue or active fragment thereof, wherein the transgenic plant is characterized by at least one of increased frequency of homologous recombination and enhanced efficacy of DNA repair compared to a corresponding non-transgenic plant. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the exogenous polynucleotide comprises a nucleic acid sequence at least 50% homologous to the nucleic acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or a fragment thereof. Each possibility represents a separate embodiment of the invention.

According to other embodiments, the exogenous polynucleotide comprises the nucleic acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8 or a fragment thereof. Each possibility represents a separate embodiment of the invention. According to certain embodiments, the RAD52 analogue fragment is devoid of a mitochondria and/or chloroplast transit peptide.

According to yet additional embodiments, the genetically modified plant comprises at least one cell having reduced expression or activity of the plant endogenous RAD52 analogue compared to a corresponding unmodified wild type plant, wherein the genetically modified plant is characterized by at least one of reduced frequency of homologous recombination and reduced efficacy of DNA repair compared to a corresponding unmodified plant. Each possibility represents a separate embodiment of the invention. Inhibiting the expression or activity of the plant RAD52 protein may be achieved by various means, all of which are explicitly encompassed within the scope of present invention. According to certain embodiments, inhibiting the plant RAD52 expression can be affected at the genomic and/or the transcript level using a variety of molecules that interfere with transcription and/or translation (e.g., antisense, siR A, Ribozyme, or DNAzyme) of the RAD52 encoding genes or transcripts. Inserting a mutation to the plant RAD52 gene, including deletions, insertions, site specific mutations, mutations mediated by zinc-finger nucleases and the like can be also used, as long as the mutation results in down-regulation of the gene expression or in non-function protein. Alternatively, expression can be inhibited at the protein level using, e.g., antagonists, enzymes that cleave the polypeptide, and the like. According to certain embodiments, the wild type unmodified RAD52 protein of the plant comprises an amino acid sequence at least 60% homologous to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7 or an active fragment thereof. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the wild type unmodified RAD52 protein of the plant comprises an amino acid sequence at least 65%, 70%>, 75%, 80%, 85%, 90% or at least 95%) homologous to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5 and 7. Each possibility represents a separate embodiment of the present invention.

According to other embodiments, the wild type unmodified RAD52 encoding gene or transcript of the plant comprises a nucleic acid sequence at least 50%> homologous to a polynucleotide having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6 and 8. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the wild type unmodified RAD52 encoding gene or transcript of the plant comprises a nucleic acid sequence at least 55%, 60%, 65%, 70%, 75%), 80%), 85%), 90% or at least 95% homologous to a polynucleotide having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6 and 8. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the genetically modified plant comprises a mutated RAD52 gene. Mutations can be enforced on a plurality of plants by any method as is known in the art, including applying mutagenic chemicals or radiation. The plurality of plants is then screened for specific mutations in the RAD52 gene by methods of molecular genetics, including molecular tilling. The plurality of plants can also be screened for a specific phenotype, e.g. for reduced frequency of homologous recombination by any suitable method as is known to a person skilled in the art.

According to additional embodiments, the genetically modified plant is a transgenic plant comprising at least one cell comprising a molecule capable of silencing the expression of the plant endogenous RAD52, the molecule is selected from the group consisting of R A interference molecule, an antisense molecule and a ribozyme-encoding molecule. The plant RAD 52 silencing molecule can be designed as is known to a person skilled in the art. According to certain embodiments, the silencing molecule comprises a polynucleotide having a nucleic acid sequence substantially complementary to a region of the expressed plant RAD52 gene.

According to some embodiments, the complementary region is of a length of 20-900 nucleotides. According to some embodiments, the complementary region is of a length of 100-400 nucleotides, or 200-300 nucleotides. According to other embodiments, the complementary region is of a length of 20-50 nucleotides, or 20-30 nucleotides.

According to certain embodiments, the silencing molecule is an antisense RNA. According to other embodiments, the silencing molecule is an RNA interference (RNAi) molecule. According to additional embodiments, the RNAi molecule is designed to produce dsRNA targeted to a plant RAD52 transcript having a nucleic acid sequence at least 60% homologous to the nucleic acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or a fragment thereof. Each possibility represents a separate embodiment of the present invention. According to certain typical embodiments, the mutation or silencing molecule is targeted to reduce the expression and/or activity of the plant endogenous RAD52 in anther or pollen cells, resulting in a male sterile plant.

Genetically modified seeds, cells and tissue cultures derived from the genetically modified plants are also encompassed within the scope of the present invention, as well as plants grown from the seeds or regenerated from the cells or tissue culture, wherein the plant have altered expression of the plant RAD52 analogue.

According to a further aspect the present invention provides a method for modulating the frequency of homologous recombination in a plant cell comprising modulating the expression of at least one RAD52 analogue protein within the plant cell. According to some embodiments, the RAD52 analogue has an amino acid sequence at least 60% homologous to the amino acid sequence as set forth in any one of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or an active fragment thereof. Each possibility represents a separate embodiment of the invention.

According to certain embodiments, the method comprises increasing the frequency of homologous recombination, said method comprising introducing into at least one plant cell an exogenous transcribable polynucleotide encoding a plant RAD52 analogue. It is to be explicitly understood that the exogenous polynucleotide can encode the endogenous plant RAD52 analogue or heterologous plant RAD52 analogue protein.

According to some embodiments, the RAD52 analogue is encoded by a polynucleotide comprising a nucleic acid sequence having at least 50%> sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or a fragment thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the RAD52 analogue is encoded by a polynucleotide comprising the nucleic acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or a fragment thereof. Each possibility represents a separate embodiment of the invention.

According to certain other embodiments, the method comprises reducing the frequency of homologous recombination, said method comprising introducing into the plant cell a molecule that silences the expression of said plant cell endogenous RAD52 analogue protein.

According to certain embodiments, the silencing molecule is selected from the group consisting of an RNA interference molecule, an antisense molecule and a ribozyme-encoding molecule. According to certain typical embodiments, the silencing molecule is targeted to a polynucleotide having a nucleic acid sequence at least 50%> homologous to the nucleic acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or a fragment thereof. Each possibility represents a separate embodiment of the present invention. According to certain typical embodiments, the silencing molecule is R Ai molecule.

According to some embodiments, the RNAi molecule is a dsRNA comprising a first nucleotide sequence of at least 25 contiguous nucleotides having at least 90%> sequence identity to the plant endogenous RAD52 transcript and a second nucleotide sequence substantially complementary to the first nucleotide sequence.

According to certain embodiment, the plant RAD52 has the nucleic acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:4 SEQ ID NO:6 and SEQ ID NO: 8 or a homolog thereof.

According to yet additional aspect, the present invention provides an isolated polypeptide derived from RAD52, said polypeptide is capable of targeting and localizing a polynucleotide to the nucleus of a plant cell, wherein said polypeptide has the amino acid sequence set forth in of SEQ ID NO:9. According to certain embodiments, the isolated polypeptide consists of SEQ ID NO:9.

Other objects, features and advantages of the present invention will become clear from the following description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows AtRAD52 genes transcripts. There are three known splice variants for AtRAD52-l and two for AtRAD52-2. AtRAD52-lB.l and AtRAD52-lB.2 are the two AtRAD52-l (Atlg71310) cDNAs with 531bp-long ORFs. AtRAD52-lA has a 498bp-long ORF. T-DNA mutant SAIL 25 H08 insertion is in the 5'UTR, 83bp 3' to the putative cDNA start, and 97bp 5' to ATG. Dashed lines indicate the location of the 385bp RNAi for silencing of AtRAD52-l transcripts through targeting of the first exon, starting at 134bp 5' to ATG, and ending at 251bp 3' to ATG. The NLS of AtRAD52-lA is marked by an open box. AtRAD52-2 (At5g47870) cDNAs are: AtRAD52-2A with a 531bp-long ORF, and AtRAD52-2B with a 600bp-long ORF. T-DNA mutant WiscDsLox303H06 insertion is in the 5'UTR 25bp 3' to the putative cDNA start, and 36bp 5' to ATG. Dashed lines indicate the location of the 383bp RNAi for silencing of AtRAD52-2 transcripts through targeting of the first exon, starting at 61bp 5' to ATG, and ending at 322bp 3' to ATG. Full transcript length are 1435bp, 2280bp, 1559bp and 1502bp for AtRAD52-lA, AtRAD52-lB.l, AtRAD52-lB.2 and AtRAD52-2B respectively. Drawings of introns, exons and UTRs are approximately to scale.

FIG. 2 demonstrates cellular localization of AtRAD52 proteins in Arabidopsis seedlings. Four-day-old Arabidopsis seedlings were transformed with Agrobacteria carrying the fusion protein constructs: FIG 2A. 52-lA-EGFP and VirE2-NLS-mRFP; FIG. 2B. 52-lB-EGFP and ScCOX4-Mito-mCherry; FIG. 2C. 52-2A-EGFP and VirE2-NLS-mRFP; Fig. 2D. 52-2B- EGFP; Fig. 2E. 52-lA-EX3-specific and VirE2-NLS-mRFP. VirE2-NLS-mRFP is a nuclear marker. ScCOX4-Mito-mCherry is a mitochondrial marker. 52-lA-EX3-specific is a short sequence that is unique to AtRAD52-lA. In each panel the first picture is of EGFP, the second is of the localization marker, the third is an overlay of the first two and the last is an image captured under visible light (DIC). All bars represent ΙΟμιη. FIG. 3 demonstrates AtRAD52 R A expression in knockout lines. R A was extracted from 8 day old seedlings. Expression level of AtRAD52-l (top) and AtRAD52-2 (bottom) was determined by real-time PCR and normalized relative to ubiquitin expression. AtRAD52-l primers were selected from a region common to all splice variants. AtRAD52-2 primers were selected from a region specific to the AtRAD52-2B splice variant (see Methods). Values represent the average of three independent repeats of the experiment. Error bars represent standard errors.

FIG. 4 demonstrates reduced fertility in atrad52 knockout lines. Plants of each genotype (mutated or silenced AtRAD52-l and AtRAD52-2) were grown in soil (n=8-18). Seeds of each plant were collected and scored and divided by the number of silks per plant. Error bars represent standard errors. ** indicates p<0.01 using ANOVA to compare WT to each genotype.

FIG. 5 demonstrates sensitivity to Mitomycin C in plants with reduced AtRAD52 gene expression. For each genotype dry weight per seedling (mg) was measured for seedlings grown in the absence of MMC or following treatment for 6 days with 1, 2, 5, 10, 20 or 30 μg/ml MMC. WT seedling were compared to each genotype using two-way ANOVA. * indicates p<0.05, ** indicates p<0.01. Each data point is an average of 3-4 repeat experiments measuring 5 seedlings each. Error bars represent standard errors.

FIG. 6 demonstrates DNA damage response and homologous recombination in plant with reduced AtRAD52 gene expression. Somatic recombination rates were evaluated using the GUS intra-chromosomal recombination assay (ICR). Results are presented per plant, for untreated plants (FIG. 6A-B: n=128 for each line; FIG. 6A C-D: WT - n=47 ; 52-2 RNAi - n =40) and per leaf, for 2μg/ml MMC-treated plants (FIG. 6A A-B: n=230 for each line; FIG. 6A C-D: WT - n=235; 52-2 RNAi - n = 302), due to higher recombination rates. WT was compared to each genotype using the Wilcoxon's non-parametric test. ** indicates p<0.01. FIG. 6A: ICR in WT vs. atrad52-l untreated plants. FIG. 6B: ICR in WT vs. atrad52-l MMC treated plants. FIG. 6C: ICR in WT vs. AtRAD52-2KNAi untreated plants. FIG. 6D: ICR in WT vs. AtRAD52-2KNAi MMC treated plants.

FIG. 7 demonstrates the ssequence similarity the Arabidopsis RAD52 type proteins and between them and representative non-plant RAD52 proteins. Vertical lines show identical aligned residues and colons show similar residues. Plus signs mark similar positions between the multiple alignment of plant RAD52 proteins and the multiple alignment of animal, fungi and protist RAD52 proteins. The N-terminal regions of the proteins are not similar to each other and are shown unaligned in lower case letters. All the sequence of the plant proteins is shown and just the N' domains of the human and yeast proteins.

FIG. 8 shows nuclear localization of AtRAD52-lA and AtRAD52-2A proteins in Arabidopsis roots, protoplasts and cotyledons. Fig. 8A: Arabidopsis roots transformed with Agrobacteria carrying the 52-lAEGFP construct. Fig. 8B: Arabidopsis cell culture protoplasts were transformed with a plasmid carrying the 52-1A-EGFP construct. Fig. 8C: Arabidopsis roots transformed with Agrobacteria carrying the EGFP construct. Fig. 8D: Arabidopsis 4 days old seedlings were transformed with Agrobacteria carrying the fusion protein constructs 52-2A-EGFP and VirE2-NLS-mRFP. Fig. 8E: Tobacco BY2 cells were transformed with Agrobacteria carrying the fusion protein constructs 52-2A-EGFP and VirE2-NLS-mRFP. All proteins are flanked by CaMV35S promoter and octopine synthase (ocs) 3'polyA signal. All bars correspond to 10 μιη.

FIG. 9 shows expression analysis of splice variants of the AtRAD52 genes. (Fig. 9A-Fig. 9E). AtRAD52 gene expression was analyzed using real-time PCR with splice-variant-specific primers sets. Expression was compared in cauline leaves, flower buds, open flowers, four- day-old seedling roots, four-day-old seedling shoots, ~3mm siliques, and ~6mm siliques. Fig. 9E: Expression of all splice variants was also compared in 16-day-old whole WT seedlings, grown in the presence or absence of 10μg/ml Mitomycin-C (MMC). Values represent the average of three independent repeats of the experiment. Error bars represent standard errors.

FIG. 10 shows analysis of TER1 expression and telomerase activity in AtRAD52-l knockdown Lines. Fig. 10A: TRF analysis of WT, atrad52-l mutant and AtRAD52-l RNAi plants. DNA was extracted from cauline leaves and digested with Msel. Digested DNAs were then run on agarose gel and blotted. The blot was hybridized with an end labeled TRF oligo 28 bp long, (T3AG3)x4. For atrad52-l mutant third generation of homozygous plants were used; for AtRAD52-l RNAi line T2 plants were used. Fig. 10B: RNA expression of TER1 was analyzed by real-time PCR on inflorescences cDNAs. TER1 expression was reduced ~6 fold in atrad52-l mutant and ~3 fold in AtRAD52-l RNAi, compared to WT. Values represent the average of three independent repeats of the experiment. Error bars represent standard errors.

FIG. 11 demonstrates that AtRAD52-lA partly complements a yeast rad52 null mutant. The three ORFs AtRAD52-lA, AtRAD52-lB, and AtRAD52-2B were cloned in pGMUlO under the inducible Gall promoter and transformed into rad52 yeast cells. Serial dilutions were plated and grown on complete SC medium containing either glucose or galactose in the presence or absence of 0.01% MMS (as indicated in top panel).

FIG. 12 shows the frequencies of Cru3 gene-targeting compared between the wild type (WT) and 35S_AtRAD52-lA_EGFP line selected for high EGFP expression (AtRAD52-lA).

^indicates that the difference was statistically significant at P= 0.0364 (FIG. 12A). A schematic illustration of the construct used is shown in FIG. 12B.

FIG. 13 demonstrates the effect of the four AtRAD52 proteins on meiotic recombination. FIG. 13A: Meiotic recombination rate in 35S-AtRAD52-lA-EGFP and 35S-AtRAD52-2A- EGFP. FIG.13B: Meiotic recombination rate in 25S-AtRAD52-lB-EGFP and 35S-atRAD52- 2B-EGFP.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses for the first time the presence of a family of RAD52 protein homologs in green plants, which affect the processes of homologous recombination and DNA repair.

The present invention now discloses two Arabidopsis RAD52 analogue genes, AtRAD52-l and AtRAD52-2, one of which, AtRAD52-l has three splice variants that encode two different proteins, and the other (AtRAD52-2) having two splice variants encoding two different proteins (Figure 1). The terms "plant RAD52" "RAD52 analogue" "plant RAD52 analogue" and "plant RAD52 protein" are used herein interchangeably, referring to the polypeptide and the polynucleotide encoding similar, or having similarity in functions, to the known yeast and mammalian RAD52 proteins, that is modulating homologous recombination and DNA repair, particularly DNA double-strand breaks (DSB) repair. The term "plant" is used herein in its broadest sense. It includes, but is not limited to, any species of woody, herbaceous, perennial or annual plant. It also refers to a plurality of plant cells that are largely differentiated into a structure that is present at any stage of a plant's development. Such structures include, but are not limited to, a root, stem, shoot, leaf, flower, petal, fruit, etc. Aaccording to one aspect, the present invention provides an isolated polynucleotide encoding a plant RAD52 protein, wherein the plant RAD52 protein has an amino acid sequence selected from the group consisting of SEQ ID NO: l (AtRAD52-lA), SEQ ID NO:3 (AtRAD52-lB) SEQ ID NO:5 (AtRAD52-2) and SEQ ID NO:7 (AtRAD52-2B), active fragments and homologs thereof. According to certain embodiments, the isolated nucleic acid comprises the nucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 SEQ ID NO:6, SEQ ID NO:8, active fragments and homologs thereof. Each possibility represents separate embodiment of the present invention. The terms "polynucleotide", "polynucleotide sequence", "nucleic acid sequence", and

"isolated polynucleotide" are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA or hybrid thereof, that is single- or double-stranded, linear or branched, and that optionally contains synthetic, non-natural or altered nucleotide bases. The terms also encompass R A/DNA hybrids.

According to certain embodiments, the AtRAD52-lA is encoded by a polynucleotide having the nucleic acid sequence set forth in any one of SEQ ID NO:2 and SEQ ID NO:30. Each possibility represents separate embodiment of the present invention. According to other embodiments, the AtRAD52-lB is encoded by a polynucleotide having the nucleic acid sequence set forth in any one of SEQ ID NO:4, SEQ ID NO:31 and SEQ ID NO:32. Each possibility represents separate embodiment of the present invention. According to additional embodiments, the AtRAD52-2A is encoded by a polynucleotide having the nucleic acid sequence set forth in SEQ ID NO:6. According to yet further embodiments, the AtRAD52-2B is encoded by a polynucleotide having the nucleic acid sequence set forth in any one of SEQ ID NO: 8 and SEQ ID NO:33. Each possibility represents separate embodiment of the present invention.

The present invention encompasses isolated or substantially purified nucleic acid or protein compositions. An "isolated" or "purified" nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Preferably, an "isolated" nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%), 5%o, (by dry weight) of contaminating protein. When the protein of the invention or biologically active portion thereof is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%>, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

Fragments and variants of the disclosed nucleotide sequences and proteins encoded thereby are also encompassed by the present invention. By "fragment" is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native RAD52 protein and hence homologous recombination and/or DNA-repair activity. As used herein, the term fragment also encompasses plat RAD52 analogues lacking the chloroplast or mitochondria transit peptide(s). The biological activity of the plant endogenous RAD52 protein is also referred to herein as RAD52 activity. Alternatively, fragments of a nucleotide sequence useful as hybridization probes generally do not encode fragment proteins retaining biological activity. Thus, fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence encoding the proteins of the invention.

The terms "variant" or "variants" as used herein refer to substantially similar sequences. For nucleotide sequences, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the RAD52 polypeptides of the invention. The term "variant" further refers to polynucleotide transcript variants resulting from alternative splicing of the plant gene. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode an RAD52 protein of the invention.

A "variant" protein as used herein refers to a protein generated by alternative splicing. The term further encompasses protein derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, homologous recombination and/or DNA-repair activity as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation.

The term "homology", as used herein, refers to a degree of sequence similarity in terms of shared amino acid or nucleotide sequences. There may be partial homology or complete homology (i.e., identity). For amino acid sequence homology amino acid similarity matrices (e.g. BLOSUM62, PAM70) may be utilized in different bioinformatics programs (e.g. BLAST, FASTA, MPsrch or Scanps) and may employ the use of the Smith and Waterman algorithms, and/or global/local or BLOCKS alignments for analysis, for example. Different results may be obtained when performing a particular search with a different matrix or with a different program. Degrees of homology for nucleotide sequences are based upon identity matches with penalties made for gaps or insertions required to optimize the alignment, as is well known in the art.

According to certain embodiments, the present invention encompasses polypeptides isolated from or present in an organism of the planta kingdom, wherein the polypeptides are at least 60% homologous to the Arabidopsis RAD52 (AtRAD52) analogues described herein. According to some embodiments, the polypeptides are at least 65%, at least 70%, at least 75%), at least 80%>, at least 85%, at least 90%>, at least 95% and more homologous to the AtRAD52 analogues described herein. Each possibility represents separate embodiments of the present invention. As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity, and thus the percentage of sequence homology. According to certain typical embodiments, the present invention encompasses plant and fungi RAD52 analogues that share at least 40% identity and 65% similarity (for RAD52 type 1) and at least 40% identity and 60% similarity (for RAD52 type 2) for alignment of at least 80 consecutive amino acids and e-values of less than 10.

The long forms of the RAD52 proteins from fungi and animals show high sequence similarity in their N-terminal domains. In contrast, the C-terminal segments of these RAD52 proteins share weak sequence similarity and contain the RAD51- and RPA-interaction regions. Moreover, the short forms of RAD52, resulting from alternative splicing, have been found to mostly contain the conserved N-terminal domain. Yeasts additionally carry the RAD59 protein, which is homologous to the N-terminal domain of RAD52 and has overlapping functions with RAD52. Bacteriophage homologs of RAD52 also only bear the RAD52 N-terminal domain. Therefore, two distinct types of RAD52 homologs, a long and short type, exist. The plant RAD52 protein sequences are similar to the short type that has only the N-terminal domain, missing the C-terminal parts of yeast RAD52 and some of the human RAD52 proteins. The similarity of plant RAD52 proteins to other RAD52 proteins is significant in terms of amino acid residues important for DNA binding, and these amino acids of human RAD52 are well conserved.

Early gene duplication within the green plant RAD52 family seems to have occurred before the emergence of seed plants. Interestingly, non seed plant representatives of Charophytes (Algae), Lycophytes (first vascular plants), Mosses and Liverworts, each have either AtRAD52-l type homologs or AtRAD52-2 type homologs (taking in account that the genomes charophytes and liverworts representatives have not been fully sequenced yet). While the separation of these two plant RAD52 types is very robust (bootstrap support value of 1000/1000), the clustering of the non-seed plant subgroups with either type is less certain. The non-seed plant members of the plant RAD52 family could represent additional types or duplication of a plant RAD52 gene that occurred very early in plant evolution but only seed plants maintained the two copies while non seed plants lost one of the two copies. Another alternative is for the duplication to have resulted from early hybridization and allopolyploidization between two species, each having one of the plant RAD52 types.

In addition to gene duplication, alternative splicing contributes to the diversity of Arabidopsis RAD52 proteins and allows for their localization within all DNA-containing cellular compartments (Figures 1 and 2). Without wishing to be bound by any specific theory or mechanism of action, the consistent sublocalization of the various AtRAD52 proteins within these organelles suggests their putative role in maintaining nuclear and organellar genomes. Mitochondrial localization of AtRAD52-lB implies a functional role similar to the role of the yeast gene MGMlOl (mitochondrial genome maintenance). The mitochondrial MGM101 is required for mitochondrial DNA replication or transmission. MGM101 features an active core with some degree of structural similarity with the RAD52 protein family. MGM101 protein contains only the RAD52 N-terminal domain.

The present invention now shows that the unique C-terminal 36 amino acids region (52- lA-ex3 -specific) of the AtRAD52-lA splice variant promoted EGFP accumulation in the nucleus. The present invention thus discloses a new nuclear localization sequence (NLS), having the amino acid sequence set forth in SEQ ID NO:9. Affecting homologous recombination within the nucleus is of significant importance in gene targeting. Fragments of the plant RAD52 analogues of the present invention, which do not include the chloroplast or mitochondrial transient peptide and thus are directed to the nucleus only, can thus be used. Without wishing to be bound by any theory or mechanism of action, the dual localization of the AtRAD52 analogues may be due to alternative translation initiation (Wamboldt Y. et al. 2009. Plant Cell 21(1), 157-167). A second ATG in AtRAD52-lA, located at positions 88-90 of AtRAD52-lA ORF (SEQ ID NO:2) could initiate a protein without the transit peptide. In AtRAD52-2A initiation of a protein without the transit peptide can starts at a GTG found in a sequence context AXXGTGG (positions 94-96 of AtRAD52-2A ORF, SEQ ID NO:6), previously disclosed as alternative translation initiation site within the Arabidopsis genome (Wamboldt et al. 2009., ibid). It is to be explicitly understood that RAD52 analogue fragments lacking the chloroplast/mitochondrial transit peptide(s) are encompassed within the scope of the present invention.

According to additional aspect, the present invention provides a genetically modified plant having altered expression of at least one RAD52 analogue. Alteration of the RAD52 analogue expression can result in at least one of reduced recombination frequency; increased recombination frequency; aberrant DNA repair or enhanced efficacy of DNA repair compared to a corresponding unmodified plant.

The term "genetically modified plant" refers to a plant comprising at least one cell genetically altered by man. The genetic modification includes modification of an endogenous gene(s), for example by introducing mutation(s) deletions, insertions, transposable element(s) and the like into an endogenous polynucleotide or gene of interest. Additionally or alternatively, the genetic modification includes transforming the organism cell with exogenous polynucleotide to produce transgenic organism. The exogenous polynucleotide can be heterologous polynucleotide or a polynucleotide endogenous to the plant.

The term "transgenic" when used in reference to a plant or seed (i.e., a "transgenic plant" or a "transgenic seed") refers to a plant or seed that contains at least one exogenous transcribeable polynucleotide in one or more of its cells. The term "transgenic plant material" refers broadly to a plant, a plant structure, a plant tissue, a plant seed or a plant cell that contains at least one exogenous polynucleotide in at least one of its cells. A "transgenic plant" and a "corresponding non transgenic plant" as used herein refer to a plant comprising at least one cell comprising an exogenous transcribeable polynucleotide and to a plant of the same type lacking said exogenous polynucleotide.

According to certain embodiments, the genetically modified plant is a transgenic plant comprising at least one cell comprising a transcribable exogenous polynucleotide encoding a plant RAD52 analogue, wherein the plant RAD52 analogue has an amino acid sequence at least 60% homologous to the amino acid sequence set forth in any one of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO: 5 and SEQ ID NO: 7 or a fragment thereof. The transgenic plant is characterized by at least one of increased frequency of homologous recombination and enhanced efficacy of DNA repair compared to a corresponding non-transgenic plant. Each possibility represents separate embodiment of the invention. An alteration in DNA repair in an organism can comprise at least one change in the

DNA of an organism or at least one cell thereof. Such changes include, but are not limited to, substitutions, additions, deletions, inversions, and other rearrangements. Typically, such an alteration in DNA repair can be determined by monitoring mutation frequency. Methods for monitoring mutation frequency are known in the art and typically involve determining whether a change has occurred in the DNA sequence of one or more genes by monitoring loss, or gain, of a particular function associated with a particular product encoded by the gene. Other methods can be employed, however, to ascertain mutation frequency at the nucleic acid level including, but not limited to, RFLP analysis, PCR, and DNA sequencing. Typically, mutation frequency is assessed by comparing the mutation frequency of a modified plant of the present invention to a control unmodified plant.

Increasing the level or activity of RAD52 analogue in a plant is expected to increase the integration of exogenous DNA through homologous recombination into specific targets within the genome. Thus, the RAD52-encoding polynucleotides of the present invention are used to increase the integration of foreign DNA into target genes within the genome. As used herein, the terms "exogenous DNA" and "exogenous polynucleotide" refer to any nucleic acid molecule that is introduced into a cell and is not present in its natural environment. It is recognized that the invention also encompasses nucleic acid molecules comprised of deoxyribonucleotides, ribonucleotides, and combination thereof. Such deoxyribonucleotides and ribonucleotides include, but not limited to, naturally occurring and synthetic form, and derivatives thereof. The exogenous polynucleotide, when referring to a gene or a transcript thereof can be a polynucleotide naturally present in the plant (endogenous polynucleotide) or a foreign polynucleotide isolated from another plant (heterologous polynucleotide). The polynucleotides of the present invention can be incorporated in a DNA construct enabling their expression in a plant cell. DNA constructs suitable for the expression of a polynucleotide within a plant cell are known to a person skilled in the art. According to one embodiment, the DNA construct comprises at least one expression regulating element selected from the group consisting of a promoter, an enhancer, an origin of replication, a transcription termination sequence, a polyadenylation signal and the like.

According to some embodiments, the promoter is selected from the group consisting of constitutive, pathogen-inducible, insect-inducible, wound-inducible, tissue-specific, and developmentally regulated promoter.

Optionally, the DNA construct further comprises a selectable marker, enabling the convenient selection of a cell/organism transformed with a polynucleotide of the invention. Additionally or alternatively, a reporter gene can be incorporated into the construct, so as to enable selection of transformed cells or organisms expressing the reporter gene.

Introducing exogenous polynucleotide into a plant cell can be performed by any method as is known to a person skilled in the art. Methods for introducing a nucleic acids sequence into a plant cell (also referred to as

"transforming a plant cell") according to the teachings of present invention are known in the art. As used herein the terms "introducing" or "introduction" and "transforming" and "transformation' "describes a process by which a foreign DNA, such as a DNA construct, enters and changes a recipient cell into a transformed, genetically modified or transgenic cell. The transformation may be stable, wherein the nucleic acid sequence is integrated into the plant genome and as such represents a stable and inherited trait, or transient, wherein the nucleic acid sequence is expressed by the cell transformed but is not integrated into the genome, and as such represents a transient trait. According to preferred embodiments the nucleic acid sequence of the present invention is stably transformed into a plant cell. There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (for example, Potrykus I. 1991. Annu Rev Plant Physiol Plant Mol Biol 42, 205-225; Shimamoto K. et al, 1989. Nature 338, 274-276). The principal methods of the stable integration of exogenous DNA into plant genomic DNA includes two main approaches:

Agrobacterium-mediatGd gene transfer: The Agrobacterium-mediatGd system includes the use of plasmid vectors that contain defined DNA segments which integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf-disc procedure, which can be performed with any tissue explant that provides a good source for initiation of whole-plant differentiation (Horsch et al., 1988. Plant Molecular Biology Manual A5, 1-9, Kluwer Academic Publishers, Dordrecht). A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially useful in the generation of transgenic dicotyledenous plants.

Direct DNA uptake: There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field, opening up mini-pores to allow DNA to enter. In microinjection, the DNA is mechanically injected directly into the cells using micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.

According to certain embodiments, transformation of the DNA constructs of the present invention into a plant cell is performed using Agrobacterium system. The regeneration, development and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach and Weissbach, In.: Methods for Plant Molecular Biology, (Eds.), 1988 Academic Press, Inc., San Diego, CA). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.

Selection of transgenic plants transformed with a nucleic acid sequence of the present invention as to provide transgenic plants characterized by increased or decresed RAD52 expression is performed employing standard methods of molecular genetics, known to a person of ordinary skill in the art. The nucleic acid sequence can further comprise a nucleic acid sequence encoding a product conferring resistance to antibiotic, and thus transgenic plants are selected according to their resistance to the antibiotic. Alternatively, the nucleic acid sequence further comprises a nucleic acid sequence encoding a product conferring resistance to an herbicide, including, but not limited to, resistant to Glufosinate ammonium.

The frequency of homologous recombination in a plant or at least one cell thereof can be reduced by reducing the expression or activity of the plant RAD52 protein. As exemplified herein below, seedling in which the AtRAD52-l gene was mutated or silenced were sensitive to Mitomycin-C (MMC) that causes damage to the DNA and showed reduced rate of an intra- chromosomal recombination rate in leaves in response to MMC. In addition, viable homozygotes of mutated AtRAD52 (atrad52-2) plants could hardly be obtained. Without wishing to be bound by any specific theory or mechanism of action, these results suggest embryo lethality of the atrad52-2 homozygotes. Further support is found in the finding that viable seeds could not be obtained from lines in which the AtRAD52-2 gene was silenced.

According to yet additional embodiments, the genetically modified plant comprises at least one cell having reduced expression and/or activity of RAD52 analogue compared to a corresponding unmodified plant, wherein the genetically modified plant is characterized by at least one of reduced frequency of homologous recombination and reduced efficacy of DNA repair compared to a corresponding unmodified plant.

According to certain embodiments, the genetically modified plant is so designed to reduce the plant endogenous RAD52 expression and/or activity in the anther and or pollen cell, wherein said genetically modified plant is male sterile. According to certain typical embodiments, the silenced RAD52 encoding gene or transcript has the nucleic acid sequence at least 50% homologous to the nucleic acid set forth in any one of SEQ ID NO: 6 and SEQ ID NO:8.

Down-regulation or inhibition of the plant RAD52 expression can be affected on the genomic and/or the transcript level using a variety of molecules that interfere with transcription and/or translation (e.g., antisense, siRNA, Ribozyme, or DNAzyme), or on the protein level using, e.g., antagonists, enzymes that cleave the polypeptide, and the like.

According to a further aspect the present invention provides a method for modulating the frequency of homologous recombination in plant cells comprising modulating the expression of a plant RAD52 protein or of a homolog thereof. According to some embodiments, the RAD52 protein has an amino acid sequence at least 60% homologous to the amino acid sequence set forth in any one of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and active fragments thereof. Each possibility represents a separate embodiment of the invention. According to certain embodiments, the method comprises increasing the frequency of homologous recombination, said method comprising introducing into at least one plant cell an exogenous polynucleotide encoding a plant RAD52 protein or a homolog thereof. The exogenous polynucleotide encoding the RAD52 protein can be the endogenous polynucleotide of the plant or a heterologous polynucleotide encoding RAD52 analogue.

According to yet another embodiment, the RAD52 analogue is encoded by a polynucleotide comprising a nucleic acid sequence having at least 50%, sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6 and SEQ ID NO:8. Each possibility represents a separate embodiment of the invention. According to certain other embodiments, the method comprises reducing the frequency of homologous recombination, said method comprising introducing into at least one plant cell a polynucleotide that silences the expression of the plant RAD52 protein.

According to certain embodiments, the silencing molecule is selected from the group consisting of RNA interference molecule, an antisense molecule and a ribozyme-encoding molecule.

The expression pattern of AtRAD52-l and AtRAD52-2 suggests that these genes are mostly non-redundant. High transcript levels of AtRAD52-2 were found in developing seeds at the torpedo and walking stick stages. AtRAD52-2 splice variants were further found to be localized in the chloroplast (AtRAD52-2A and AtRAD52-2B) and in the nucleus (mainly AtRAD52-2A). Interestingly, several genes that encode chloroplast-localized proteins have an embryo-lethal mutant phenotype. Similarly, most genes identified in a genetic screen for a seedling-lethal mutant phenotype, encode proteins predicted to have chloroplast localization. The present invention now shows that hardly any viable seeds were produced in plants homozygous to the non-functional AtRAD52-2 gene. Without wishing to be bound by any specific theory or mechanism of action, one possible interpretation of the embryo-lethality phenotype is that chloroplast genome stability maintenance is essential to enable the chloroplast to carry its metabolic functions during seed development. Directing silencing molecule targeted to RAD52-2 to anther or pollen cell can thus be employed for the production of male sterile plants, which are highly desirable in plant breeding. The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention. EXAMPLES

Experimental procedures

Bioinformatics and Phylo genetic Analysis

Sequence searches were performed on the NCBI databases using the BLAST and PSI- BLAST programs. The queried databases were of protein, genomic, and transcribed (EST) sequences from the NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and JGI (http :// genome.i gi- psf.org). ESTs were assembled using the CAP3 program (Huang X. and Madan A, 1999. Genome Res 9, 868-877). Multiple alignments were found using PSI-BLAST, BlockMaker (Henikoff S. et al, 1995. Gene 163, GC17-26), Macaw (Schuler G.D. et al, 1991. Proteins 9, 180-190), and FSA (Bradley R.K. et al, 2009. Fast statistical alignment. PLoS Comput Biol 5, el000392) programs. Sequence logos (Schneider T.D. and Stephens R.M, 1990. Nucleic Acids Res 18, 6097-6100) were calculated as previously described (Henikoff S. et al, 1995. ibid). The conservation of the alignment positions is taken from the position specific scoring matrix (PSSM) of protein multiple alignments. The conservation values are the information content of each position in bits, corresponding to the total height of each column of the sequence logos. Multiple alignment to multiple alignment comparisons were performed using the LAMA (Frenkel-Morgenstern M. et al, 2005. Bioinformatics 21, 2950-2956) and Compass (Sadreyev R. and Grishin N., 2003. J Mol Biol 326, 317-336) programs. Structure models were calculated on the LOMETS ( Wu S. and Zhang Y. 2007. Nucleic Acids Res 35, 3375-3382), Phyre (Kelley L.A. and SternbergM/J., 2009. Nat Protoc 4, 363-371), and mGenThreader (McGuffin L.J. and Jones D.T., 2003. Bioinformatics 19, 874-881) servers. Several equally stable models, based on the known structure of human RAD52 N-terminal domain were found. The model used in this work was calculated on the LOMETS server by the HHsearch (Soding J. 2005. Bioinformatics 21, 951-960) program. PyMol (version 0.99rc6 from DeLano Scientific) was used to examine protein structures, calculate their approximate charge distribution, map the sequence conservation on the structure, and generate figures. Phylogenetic trees were calculated in PHYML (Guindon S. and Gascuel O., 2003. Syst Biol 52, 696-704.), version 2.4.4, with four substitution rate categories and other default parameters to estimate the Ts/Tv ratio, gamma shape parameter and invariant proportion. These parameters were then used with 1000 bootstrap replicates. The dendogram was outgroup rooted from the position of animal, fungal, and protist RAD52 sequences that were added to the alignment. Mutants

T-DNA tagged mutants were obtained from the Arabidopsis Biological Resource Center (Alonso J.M. et al, 2003. Science 301, 653-657). Border cloning and sequencing for each mutant homozygous line provided the corresponding gene's sequence from both ends of the T-DNA. atrad52-l features a left border (LB) at the 5' and right border (RB) at the 3', and atrad52-2 has LB at both ends of the insertion. In the atrad52-l mutant SAIL 25 H08 T- DNA insertion is in the 5'UTR, 83bp 3' to the putative cDNA start, and 97bp 5' to ATG (Figure 3). The atrad52-2 mutant WiscDsLox303H06 T-DNA insertion is in the 5' UTR, 25bp 3' to the putative cDNA start, and 36bp 5' to ATG. cDNAs

AtRAD52-l (Atlg71310) cDNAs sources: Atlg71310.1 (AtRAD52-lB. l) 531bp-long ORF: NM 105800: SQ036a05F (Kazusa Japan). Atlg71310.2 (AtRAD52-lB.2) 531bp-long ORF: NM 179545: RAFL11-06-122 (Riken Japan) Atlg71310.3 (AtRAD52-lA) 498bp-long ORF: NM 202394: BX816488 ((Institut National de la Recherche Agronomique,France). The three publicly available cDNA clones of AtRAD52-l splice variants were sequenced, and their sequences were identical to the ones published in public databases. To clone AtRAD52- 2A cDNA, we used RT-PCR on WT Arabidopsis Columbia four-day-old seedling roots cDNA with the following primers:

AR52-2 RI F gtcgacgaattcATGGCTTTGCAAGTGCAGC (SEQ ID NO: 10) AR522A_Xho_R ctcgagTCACACACAACTACACCC (SEQ ID NO: 11)

To clone AtRAD52-2B cDNA, we used RT-PCR on WT Arabidopsis Columbia inflorescences cDNA (Sequence identical to NM_ 124161 ORF) with the following primers:

AR52-2 RI F gtcgacgaattcATGGCTTTGCAAGTGCAGC (SEQ ID NO: 12)

AR52-2_Xba_R GtctagaCTATTCGTGGTATAGATACAAGCCG (SEQ ID NO: 13). Genotoxicity Assay for Yeast Cells

The yeast MK166-52 rad52 strain (kindly provided by Martin Kupiec (Liefshitz B. et al., 1995. Genetics 140, 1199-1211) was transformed with AtRAD52 homologs that were cloned into pGMUlO (Iha H. and Tsurugi K., 1998. Biotechniques 25, 936-938) under the inducible Gall promoter. The strain BY4742 was used as the WT control. MMS sensitivity was tested by growing yeast cells to stationary phase in SD-Leu-Ura (SD-Trp for WT) medium and then diluted and grown again to logarithmic phase in liquid SC-Ura medium supplemented with either 2% glucose or galactose and 1% raffmose. Yeast were counted and plated in serial dilutions on SC + glucose with or without MMS (0.01%) and on SC + galactose with or without MMS (0.01%). Plates were incubated at 30°C for 5 days.

Cellular RAD52 Localization

Four day old Arabidopsis seedlings were transformed with Agrobacteria carrying fusion protein constructs as previously described in (Li J.F. et al, 2009. Plant Methods 5, 6). Arabidopsis roots were transformed with Agrobacteria carrying fusion protein constructs using the method described in Gelvin (Gelvin S. B., 2006. Methods Mol Biol 343, 105-113). Transformation of Arabidopsis cultured cells protoplasts was performed essentially as described in Yoo et al. (Yoo S.D. et al, 2007. Nat Protoc 2, 1565-1572). Tobacco (Nicotiana iahacum) BY2 cells at 20% cells per volume stage were mixed with 1 /80 volume of agrobacteria carrying fusion protein constructs in logarithmic phase and visualized 3 days after transformation. At RAD 52- 1 and At RAD 52-2 ORFs were fused to EGFP at the C-terminal end by replacing of the stop codon with an Ncol site. ScCOX4-Mito-mCherry is the mitochondrial targeting signal of yeast ScCOX4 fused to mCherry mt-rk CD3-991 (Nelson B.K. et al, 2007. Plant J 51, 1126-1136). The VirE2-NL S -mRFP consisting of four repeats of the minimal 134bp VirE2 NLS fused to mRFP was a kind gift from Prof. Yuval Eshed (Alvarez J.P. et al, 2009. Plant Cell 21, 1373-1393). All proteins aside from ScCOX4-Mito- mCherry were flanked by the CaMV35S promoter and the octopine synthase (ocs) 3'polyA signal. ScCOX4-Mito-mCherry was flanked by a double CaMV35S promoter and the nopaline synthase (nos) 3'polyA signal. Cellular localization was analyzed using a laser confocal microscope Olympus 1X81 FV1000 Spectral, equipped with an FV1000 UPLAPO 60xO NA: 1.35 objective lens. EGFP images were captured using Argon laser (excitation: 488nm; emission 500-545nm interval). mRFP and mCherry labeled proteins were viewed using Diode laser (excitation: 559nm and emission through 575-620nm filter). Chlorophyll was detected using Diode laser (excitation: 638nm; emission 655-755nm filter). 4',6- diamidino-2-phenylindole (DAPI) was viewed using Diode laser (excitation: 405nm; emission through 415-435nm filter). Imaging was performed in a line sequential mode.

RNAi Lines

A 385bp fragment was used for RNAi silencing of AtRAD52-l transcripts, which target the first exon, starting 134-bp 5' to ATG and ending 251-bp 3' to ATG. A 383-bp fragment was used for RNAi silencing of AtRAD52-2 transcript, which target the first exon, starting at 61 -bp 5' to ATG and ending at 322-bp 3' to ATG. RNAi regions for each gene were cloned in the pKANNIBAL vector in the order 5' CaMV35S promoter, sense orientation, intron, antisense orientation and 3' octopine synthase (OCS) 3'polyA signal (Wesley S.V. et al, 2001. Plant J 27, 581-590). Transcript Analysis by Real-Time RT-PCR

Total RNA was extracted using Tri reagent (Molecular Research Center, Inc.). Three repeat experiments of eight 8-day-old seedlings of each mutant and RNAi line were evaluated for changes in AtRAD52 genes expression cDNA was synthesized using Superscript II RNase H- Reverse Transcriptase (Invitrogen Life Technologies) and oligo (dTig) primer. Reactions for quantitative realtime RT-PCR on the cDNA were performed using Applied Biosystems Power SYBR Green and run on an Applied Biosystems 7300 cycler. Reactions for each gene in each cDNA sample were repeated independently at least three times. Ubiquitin (At5g25760) was used as a reference for cDNA amount. Quantification of each gene was performed using Applied Biosystems 7300 software. Relative expression of a gene in a certain sample was initially obtained by dividing the gene expression level (in arbitrary units) by the ubiquitin level (in arbitrary units). Relative expression units are shown by setting the sample with the lowest or highest expression at a value of 1. The following primers were used for analysis of AtRAD52 genes expression in lines with reduced expression (Figure 3):

AtRAD52-l PCR primers: 52-lCF - TCGGATCATGAATATGCATGCT (SEQ ID NO: 14); 52-lCR - AGTCACACGATAAGCCACAGTAACA (SEQ ID NO: 15), the primers are in a region 3' to AXRAD52-1 RNAi that is common to all AtRAD52-l splice variants.

AtRAD52-2 PCR primers: 52-2F - TGGTGAGGCACATCGTGAAT (SEQ ID NO: 16); 52-2C - TGCTCTGCAGAATGCTATTTCC (SEQ ID NO: 17) the primers are in a region 3' to AtRAD52-2 RNAi.

Ubiquitin PCR primers: Ubiquitin C-F AGCGCGACTGTTTAAAGAATACA (SEQ ID NO: 18); Ubiquitin C-R TTGTGCCATTGAATTGAACCC (SEQ ID NO: 19).

The following primers were used for analysis of specific AtRAD52 splice variants in different tissues and for evaluating plant responses to MMC treatment.

AtRAD52-lA: 521AUF- TCTTTGCCTTCTTGCCTCAGA (SEQ ID NO:20);

521 A UR- ATTCCTTGACGGGTTTCATCAT (SEQ ID NO:21).

AtRAD52-lB.l: 521B1UF- ATGAGTGCGCGAAGACAAGA (SEQ ID NO:22);

521B1UR- AAAAGC CG AGC C AGT AAGC A (SEQ ID NO:23).

AtRAD52-lB.2 521B2UF -TCAGCTGATATTGATCGCTTTATGA (SEQ ID NO:24); 521B2UR-GTTACAGAAATGCAACAAAGAGTCCT (SEQ ID NO:25). AtRAD52-2A: 522AF-TTGGTCATCTATGGCAATGCC (SEQ ID NO:26); 522AR-TACAACTTCAACAACCAATCCAACTC (SEQ ID NO:27). AtRAD52-2B primers were as indicated above. RNA was extracted from inflorescences for analysis of TER1 expression in AtRAD52-l knockdown lines. cDNA was synthesized using Superscript III, as described by Cifuentes-Rojas et al. (Cifuentes-Rojas C. et al, 2011. Proc Natl Acad Sci U S A 108, 73-78.). The following primers were used for the PCR:

TER 1 UF-TTCTC AGGC AC ATTGTGAATCG (SEQ ID NO:28);

TER1UR-ATGTCAAAACCAAAACCCAACAAC (SEQ ID NO:29).

Terminal Restriction Fragment (TRF) Analysis

TRF was assayed as described by Cifuentes-Rojas et al. (Cifuentes-Rojas et al, 2011, ibid)

Mitomycin C

Mitomycin C assay was performed as previously described (Hartung F. et al, 2007. Proc Natl Acad Sci U S A 104, 18836-18841) with freshly prepared MitomycinC (MMC) solutions (Sigma Cat# M4287). Intra chromosomal recombination (ICR) assay

The GUS tester line (Swoboda P. et al, 1994. Embo J 13, 484-48) was crossed with the atrad52-l Arabidopsis mutant. F3 seeds from plants homozygous for both the GUS recombination substrate and for the atrad52-l were tested. The AtRAD52-2 RNAi construct was transformed to the GUS tester line. T2 seeds from a line that showed 5-fold reduction of AtRAD52-2 expression were tested. Seeds were plated on solid 1/2 MS medium. At eight days, seedlings were transferred to liquid ½ MS medium. After nine days, MMC was added to a final concentration of 2μg/ml.

Medium was added to the same volume as MMC in cases of "untreated" seedlings. At 16 days, seedlings were stained for GUS activity. The number of blue spots or sectors on each seedling was visually determined using a light microscope.

Seed Counting

Plants were grown and seeds were collected from the beginning of maturation until the whole plant dried. Seed number was calculated based on 100 seeds weight.

Accession Numbers

Sequence data can be found in the EMBL/GenBank data libraries under the following accession numbers. Atlg71310.1 (AtRAD52-lB ORF): NM 105800: SQ036a05F (Kazusa Japan), Atlg71310.2 (AtRAD52-lB ORF): NM 179545: RAFL11-06-122 (Riken Japan), Atlg71310.3 (AtRAD52-lA ORF): NM_202394: BX816488 (INRA France). At5g47870 (AtRAD52-2).

Example 1: Higher plants include a family of RAD 52 homologs encoding several genes and splicing isoforms Plant RAD52 homologs were first identified in the Arabidopsis genome, using PSI

BLAST program using the yeast RAD52 protein as the query. A putative protein was detected, (Atlg71310, accession number NP_849876.1), with an e-value of le ⁰⁴, and 30.7% identity to the yeast RAD52 56-176 N-terminal residues. Next, multiple sequence alignments (MSAs) of land plants homologous to the Arabidopsis RAD52 like protein were constructed. These plant proteins are about 170-220 residues long, with weakly conserved N-termini and a well-conserved 129 residues region at the central and C-terminal parts. This latter region was found significantly similar with an e-value of 7.8 10^"6 (Compass program,(Sadreyev R. and Grishin N., 2003. J Mol Biol 326, 317-336) to MSAs of RAD52 proteins from animals, fungi, and lower eukaryotes, in the RAD52 catalytic domains for homologous pairing and multimerization (Figure 7). Of note, human residues found to play an important role in DNA binding, tend to be conserved across kingdoms, including in plants.

Plant RAD52 homologs were identified by careful and thorough sequence analysis of various available plant data sources, covering proteins, expressed sequence tags (ESTs), transcriptomes and genome data. Full and partial protein sequences were assembled from diverse land plants, from liverworts and mosses to flowering plants, and in charophyte unicellular green algae . The plant RAD52 homolog family was categorized into two subtypes, each of whose members were found in gymnosperms, monocots dicots and liverworts. Some lineages have only one homolog type, e.g. mosses with only type 2, and charophytes with only type 1. The sequence similarity between the two RAD52 homolog types is was typically 40-67% across the C-terminal, two thirds (120-150 amino acids) of the proteins (Figure 7). The presence of the two homolog types suggests a gene duplication that occurred as early as the appearance of non- vascular land plants. Later duplications were apparent in specific lineages. These duplicates are separate genes within the same strain rather than different alleles in the same strain or in different strains of the same species. This was verified by finding the duplicates in different genomic contexts, by finding several duplicates in single strains, and by finding common duplicates in several related species, as in the case of the grasses which include two type-2 RAD52 homologs. Mosses, represented by two very well sequenced species (Physcomitrella patens (Rensing S.A. et al, 2008. Science 319, 64-69) and Syntrichia ruralis (Oliver M.J. et al., 2004. BMC Genomics 5, 89) seem to include only type-2 RAD52 homologs. P. patens has two type-2 PvAD52 homologs that apparently underwent duplication within the mosses lineage.

While almost all duplications were in type-2 RAD52 homologs, maize features two type-1 paralogs on chromosomes 3 and 8. Sequences of type-1 RAD52 homologs from transcribed genes were found in the Chara vulgaris and Spirogyra pratensis charophyte unicellular green algae species (Timme R.E. and Delwiche C.F., 2010. BMC Plant Biol 10, 96). Lack of identifiable type-2 RAD52 homologs in the charophytes available expressed gene data, could be due to their absence from this lineage or to specific or low expression of their genes. In either case, the type-1 and type-2 duplication apparently predates the divergence of the charophyte lineage. No RAD52 homologs were found in extensive data available for other major lineages of algae, including chlorophyte green algae, rhodophyte red algae, haptophytes algae, and heterokont (stramenopiles; including brown algae, diatoms, and oomycetes).

The described sequence similarity between plant and other RAD52 proteins allowed us to model the structure of plant RAD52 homologs on the determined structures of the N- terminal domain of human RAD52 (Kagawa W. et al, 2002. Mol Cell 10, 359-371; Singleton M.R. et al, 2002. Proc Natl Acad Sci U S A 99, 13492-13497). As expected by the relatively long corresponding regions and few insertion/deletion points (Figure 7) the same topology and structure features of human RAD52 were found to be present in the models of Arabidopsis RAD52 homologs. The residues corresponding to the two known RAD52 DNA binding sites (Kagawa et al, 2002 ibid; Singleton et al, 2002 ibid) are the most conserved regions in the plant proteins, and form similar sites, including both the positively charged groove and the second DNA binding site. The alpha helix of the RAD52 stem region (Residues 145-163 in Atlg71310, corresponding to residues 159-134 in human RAD52) has a repetitive pattern of sequence conservation. Residues facing and interacting with the stem region beta sheet were highly conserved in the plant RAD52 homologs while residues facing the predicted ssDNA binding site were not conserved. These features reinforce the functional homology between the plant RAD52 homolog family and the known RAD52 proteins, suggest similar function for these two families, and should assist molecular studies of plant RAD52 proteins.

AtRAD52-l (Atlg71310) and AtRAD52-2 (At5g47870), the two identified Arabidopsis RAD 52 homologs included three splice variants for AtRAD52-l, encoding open reading frames (ORFs), AtRAD52-lA and AtRAD52-lB. Two splice variants were found for AtRAD52-2, encoding ORFs AtRAD52-2A and AtRAD52-2B (Figure 1). The Atlg71310.3 (AtRAD52-lA ORF) splice variant bore three exons, while splice variants Atlg71310.1 and Atlg71310.2 both encoded the same ORF (AtRAD52-lB), and each feature four exons, but differ in the number of introns and in the length of the 3' UTR. The AtRAD52-2A transcript had at least two exons, and the A tRAD52-2B transcript had three exons (Figure 1). Example 2: AtRAD52 proteins localize in the nucleus, mitochondria and chloroplasts

Each of the AtRAD52 ORFs was fused to EGFP and the resulting constructs were transiently expressed in Arabidopsis seedlings, roots, and cell culture protoplasts. Protein localization was determined in cells using confocal microscopy to detect co-localization of the EGFP fluorescence and cellular markers (Figure 2). AtRAD52-lA was found in the three cell types (Fig 2A, and Figure 8B) to co-localize with the VirE2-NLS-mRFP and DAPI, the positive controls for nuclear localization.

AtRAD52-lA was homogenously expressed throughout the nucleus, with the exception of the nucleolus. Note that EGFP alone does not localize to the nucleus (Figure 8C). AtRAD52-lB co-localized with the mitochondrial ScCOX4-Mito-mCherry (Figure 2B). Unlike AtRAD52-lA, AtRAD52-2A expression was only apparent in foci at the periphery of the nucleus and chloroplast (Figure 2C and Figure 8D). AtRAD52-2B demonstrated a punctate expression pattern within the chloroplast (Figure 2D).

The differential localization between AtRAD52-l-A and AtRAD52-lB might be determined by their different C-terminal regions generated by alternative splicing (Figure 1). The unique 36 amino acids C-terminal region of AtRAD52-l-A (VFPFLLFQCYGLIWLAFSSSRLLVEFFAFLPQKLQI SEQ ID NO:9), named herein 52- 1A-EX3 -specific, was therefore fused to EGFP and found to co-localize with the VirE2-NLS- mRFP, the positive control for nuclear localization (Figure 2E). The 52-lA-EX3-specific sequence was found to lack similarity to known nuclear localization signals (NLS). The present invention thus discloses a novel sequence that directs nuclear localization in Arabidopsis. In summary, the AtRAD52 homologs are localized in all the plant DNA- containing compartments.

Example 3: expression of AtRAD52-l and AtRAD52-2 in wild type, mutant and RNAi Arabidopsis lines AtRAD52 expression was analyzed using real-time PCR with splice-variant-specific primers sets (Figure 9). Expression was compared between cauline leaves, flower buds, open flowers, four-day-old seedling roots, four-day-old seedling shoots, ~3mm siliques, and ~6mm siliques. AtRAD52-lA transcription level was similar in all these tissues, with approximately 1.5-fold higher expression in seedling roots (Figure 9A). AtRAD52 transcripts, except AtRAD52-lA, showed a marked increase of ~ 100-fold in plant tissues of four-day-old seedlings shoots and roots (Figure 9B-E), when compared to other tested tissues. Expression of each splice variant was also compared in 16-day-old whole wild type (WT) seedlings grown in the presence or absence of 10μg/ml Mitomycin-C (MMC) (Figure 9F). Treatment with MMC was 6 days long, from day nine to day 16, and was part of the same experiment described in Figure 5. The changes observed at this stage were relatively minor even if in some cases statistically significant.

Mutant and R Ai lines with altered AtRAD52-l and AtRAD52-2 expression were generated and characterized in order to study the functional roles of these genes. A homozygous line was prepared for the atrad52-l SAIL 25 H08 T-DNA insertion allele, giving rise to atrad52-l; and for the atrad52-2 WiscDsLox303H06 T-DNA insertion allele, giving rise to atrad52-2. Only two atrad52-2 homozygotes were found among the 46 progeny obtained from heterozygote AtRAD52/atrad52-2 plants, a ratio significantly lower than that expected via Mendelian inheritance (X² P value=0.0012). The atrad52-l homozygotes yielded the expected 1 :3 ratio.

RNAi lines were constructed, for repression of AtRAD52-l and AtRAD52-2 transcripts (see Materials and Methods). Three single-locus T-DNA lines of each RNAi construct were selected for further analyses. The expression of the AtRAD52 genes transcripts was determined by Real-Time PCR on mRNA isolated from seedlings of WT, mutant and RNAi lines (Figure 3). Expression analysis of the AtRAD52-l transcripts in the various lines, detected that atrad52-l exhibited almost null expression compared to WT; AtRAD52-l RNAi lines (52-1 RNAi) showed reduced expression ranging from moderate to ~ 60,000-fold less than WT (see Figure 3, line 52-1 RNAi3). atrad52-l and line 52-1 RNAi3 were selected for further characterization of the effect of reduced AtRAD52-l expression. Upon RNAi inhibition of AtRad52-2, AtRAD52-l exhibited slightly higher transcript levels than WT; this was not significant but was consistent in all RNAi lines, suggesting the possibility of compensation. Similarly, expression analysis of the AtRAD52-2 transcript in the various lines showed that the atrad52-l and AtRAD52-l RNAi lines had slightly more AtRAD52-2 transcript than WT. The expression of the AtRAD52-2 transcript in the atrad52-2 mutant was reduced by ~2-fold. AtRAd52-2 RNAi (52-2 RNAi) lines showed a reduction in expression ranging from 5-12-fold, compared to WT (Figure 3). atrad52-2 and line 52-2 RNAi8 were selected for further characterization of the AtRAD52-2 reduced-expression phenotype. The atrad52-l mutant and 52-2 RNAi lines were combined for a most effective double knockdown.

It was recently described that the TER1 transcript overlaps with the first two exons and introns oiAtRAD52-l (Cifuentes-Rojas C. et al, 2011. Proc Natl Acad Sci U.S.A 108, 73-78). TER1 is the RNA subunit of telomerase, and serves as a template for telomeric DNA addition. Without wishing to be bound by any specific theory or mechanism of action, TER1 transcript levels and the consequential telomerase activity, could potentially be affected by the atrad52- 1 mutant and the AtRAD52-l RNAi, and influence the phenotypic manifestation of these lines. For this reason, TER1 transcript levels were analyzed in inflorescences, known to highly express TER1. A 6-fold and 3-fold decrease in TER1 expression was observed in the atrad52- 1 mutant and the AtRAD52-l RNAi, respectively, when compared to WT (Figure 10). However, Terminal Restriction Fragment (TRF) analysis was also performed and no decrease in telomere length was detected in either line, when compared to WT (Figure 10).

Example 4: phenotypic analysis of atrad52-l and atrad52-2 mutants and RNAi lines Significant reductions in overall fertility, expressed as the number of seeds per silique, correlated with reduced AtRAD52 genes expression in the tested mutant and RNAi lines (Figure 4). However, no additive effect on fertility was observed upon combination of atrad52-l and AtRAD52-2 RNAi, when compared to each of these lines separately. The measured reduction in seed number per silique in the various lines with reduced AtRAD52 genes expression, suggest the involvement of AtRAD52-l and AtRAD52-2 in the viability of both somatic and meiotic cell types.

Example 5: Role of AtRAD52-l and AtRAD52-2 in DNA damage response and homologous recombination

A series of assays were then performed to evaluate the expected role of AtRAD52-l and AtRAD52-2 in plant DNA recombination and DNA damage response. In complement models, AtRAD52-lA complemented the yeast rad52 MMS sensitive phenotype, to a lesser extent than ScRAD52 (Figure 11). Complementation was only partial, but reproducible, suggesting that a plant AtRAD52 proteins retained DNA repairing roles similar to those performed by the yeast RAD52 homolog. The involvement of AtRAD52 proteins in DNA damage repair was then tested following the MMC-induced DNA damage, by monitoring seedling growth. Inter-strand cross-links mediated by MMC can lead to DSBs, which, in turn, can be repaired by homologous recombination. Both the atrad52-l mutant and AtRAD52-l RNAi lines showed a significant reduction in seedling growth following MMC treatment (Figure 5A), compared to WT. Similar reductions in growth rates were also observed in the atrad52-2 mutant and AtRAD52- 2 RNAi lines (Figure 5B). Sensitivity to MMC treatment was not additive in the atrad52-l and AtRAD52-2 RNAi combination (Figure 5C), in agreement with the observed combination fertility phenotype.

Somatic recombination were assessed in the atrad52-l mutant and AtRAD52-2 RNAi lines using an intra-chromosomal recombination (ICR) assay, with a reporter transgene consisting of two overlapping fragments of the GUS (uidA) gene separated by a hygromycin selectable marker (Swoboda P. et al., 1994. ibid). The atrad52-l mutant plants and GUS recombination reporter plants were crossed, and the frequency of recombination events was monitored in F3 progeny plants homozygous for both the recombination reporter and the atrad52-l mutation (see Methods). Somatic ICR in WT versus mutant under normal or MMC- induced conditions was determined. The AtRAD52-2 RNAi construct was directly transformed into the ICR line, and a line demonstrating a ~5-fold reduction in AtRAD52-2 RNA was selected for further analysis in T2 progeny plants. Somatic ICR frequencies in WT versus mutant lines, under normal or MMC-induced conditions was determined. No difference in ICR rates was detected in the atrad52-l mutant under normal conditions (Figure 6A), however, when seedlings were treated with MMC, ICR rates were significantly reduced in the mutant compared to WT (Figure 6B). In the AtRAD52-2 RNAi line, a significant reduction in ICR rates, compared to WT, was detected under both normal and MMC treatment conditions (Figure 6C and 6D, respectively). Both the somatic homologous recombination phenotype and the MMC sensitivity of the AtRAD52 mutants and RNAi lines are indicative of defective repair pathways.

The effect of the Arabidopsis nuclear splice variant AtRAD52-lA on homologous recombination was further analyzed by assessment of gene-targeting in Arabidopsis plants overexpressing AtRAD52-lA. The gene-targeting assay was performed as in Even-Faitelson L et al. (Plant J. 2011 Dec;68(5):929-37). AtRAD52-lA ORF was flanked by the CaMV35S promoter and the octopine synthase 3' polyA signal. This cassette was cloned into the pMLBArt binary vector, which contains a gene conferring to plants resistance to the non- selective herbicide glufosinate-ammonium (BASTA®). A single insertion line that gives high EGFP fluorescence indicating high AtRAD52-lA-EGFP expression was used in these experiments.

Overexpression of AtRAD52-lA-EGFP increased the gene -targeting by about 10 fold. Frequencies of Cru3 gene-targeting were compared between the wild type (WT) and 35S_AtRAD52-lA_EGFP line selected for high EGFP expression (AtRAD52-lA). The gene- targeting frequency was approximately 10-fold higher in 35S_AtRAD52-lA_EGFP than in the wild type (WT) (Table 1 and Figure 12). This difference was statistically significant: P= 0.0364. Table 1 : Effect of Arabidopsis RAD52 variant AtRAD52-l A on gene targeting

The effect of the four AtRAD52 proteins on meiotic recombination was also examined. Lines expressing 35S-AtRAD52 proteins with c-terminal EGFP fusion were selected for high EGFP expression and then crossed with a meiotic tester line harboring two fluorescent seed- coat markers. Progeny were analyzed for meiotic recombination rates between these markers. Recombination rates were compared to WT crossed with the meiotic tester line.

The constructs used were:

35Spromoter-AtRAD52-lA-EGFP-OCSterminator - high expressing line, same as used for gene-targeting experiment above. 35Spromoter-AtRAD52-lB-EGFP-OCSterminator - high expressing lines were not obtained. Without wishing to be bound by any specific theory or mechanism of action, high expression of this RAD-52 variant resulted in non-viable lines. Accordingly, line expressing low levels of AtRAD52-lA was used.

35Spromoter-AtRAD52-2A-EGFP-OCSterminator - as for the AtRAD52-lA line, high expressing lines were not obtained. Low expressing line was used.

35Spromoter-AtRAD52-2B-EGFP-OCSterminator - high expressing line was obtained and used.

Over expression AtRAD-52-lA resulted in an increase in the meiotic recombination rate by about 14% compared to wild type (Figure 13). Such increase was not detected in the lines expressing other RAD52 variant. This increase may indicate the involvement of AtRAD-52-1 A in the meiotic recombination process. The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

1. A genetically modified plant having altered expression of at least one plant RAD52 analogue compared to a corresponding unmodified plant, wherein the plant RAD52 analogue has an amino acid sequence at least 60% homologous to any one of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or an active fragment thereof.

2. The genetically modified plant of claim 1, said plant is a transgenic plant comprising at least one cell comprising a transcribable exogenous polynucleotide encoding the at least one plant RAD52 analogue or active fragment thereof, wherein said transgenic plant is characterized by increased frequency of homologous recombination compared to a corresponding non-transgenic plant.

3. The genetically modified plant of claim 1, said plant is a transgenic plant comprising at least one cell comprising a transcribable exogenous polynucleotide encoding the at least one plant RAD52 analogue, wherein said transgenic plant is characterized by enhanced efficacy of DNA repair compared to a corresponding non-transgenic plant.

4. The genetically modified plant of any one of claims 2-3, wherein the transcribable exogenous polynucleotide comprises a nucleic acid sequence at least 50% homologous to the nucleic acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or a fragment thereof.

5. The genetically modified plant claim 4, wherein the transcribable exogenous polynucleotide comprises the nucleic acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or a fragment thereof.

6. The genetically modified plant of any one of claims 2-3, wherein the RAD52 analogue fragment is lacking a mitochondria and/or chloroplast transit peptide.

7. The genetically modified plant of claim 1, said plant comprises at least one cell having reduced expression or activity of said plant endogenous RAD52 analogue, wherein said genetically modified plant is characterized by reduced frequency of homologous recombination compared to a corresponding unmodified plant.

8. The genetically modified plant of claim 1, said plant comprises at least one cell having reduced expression or activity of said plant endogenous RAD52 analogue, wherein said genetically modified plant is characterized by reduced efficacy of DNA repair compared to a corresponding unmodified plant.

9. The genetically modified plant of any one of claims 7-8, wherein the polynucleotide encoding said plant endogenous RAD52 analogue is at least 50% homologous to a polynucleotide comprising the nucleic acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or a fragment thereof

10. The genetically modified plant of claim 7, wherein the polynucleotide encoding said plant endogenous RAD52 analogue comprises at least one mutation, wherein the mutation results in reduced expression or activity of said plant endogenous RAD52 analogue.

11. The genetically modified plant of claim 9, said plant is a transgenic plant comprising at least one plant cell comprising a silencing molecule capable of silencing the expression of the polynucleotide encoding said plant endogenous RAD52 analogue.

12. The genetically modified plant of claim 11, wherein the silencing molecule is a polynucleotide having a nucleic acid sequence substantially complementary to a region in the polynucleotide encoding said plant endogenous RAD52 analogue .

13. The genetically modified plant of claim 12, wherein the silencing molecule is an R A interference (R Ai) molecule.

14. The genetically modified plant of claim 13, wherein the RNAi molecule is designed to produce dsRNA targeted to the polynucleotide encoding said plant endogenous RAD52 analogue .

15. The genetically modified plant of claim 11, wherein the silencing molecule is expressed in anther or pollen cell, thereby conferring male sterility on said plant.

16. A seed of the genetically modified plant of claim 1, wherein a plant grown from said seed has altered expression of a plant RAD52 analogue compared to a plant grown from a corresponding seed of unmodified plant.

17. A tissue culture comprising at least one genetically modified cell of the plant of claim 1 or a protoplast derived therefrom, wherein a plant regenerated from said tissue culture has an altered expression of a plant RAD52 analogue compared to a corresponding unmodified plant.

18. A plant regenerated from the tissue culture of claim 17.

19. A method for modulating the frequency of homologous recombination in a plant cell comprising modulating the expression of at least one RAD52 analogue protein within the plant cell.

20. The method of claim 19 wherein, the RAD52 protein analogue has an amino acid sequence at least 60% homologous to the amino acid sequence set forth in any one of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or an active fragment thereof.

21. The method of claim 19, said method comprises introducing into the plant cell an exogenous transcribable polynucleotide encoding a plant RAD52 analogue or an active fragment thereof, thereby increasing the frequency of homologous recombination.

22. The method of claim 21, wherein the polynucleotide encoding the plant RAD52 analogue has a nucleic acid sequence at least 50% homologous to any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8 or a fragment thereof.

23. The method of claim 22, wherein the polynucleotide encoding the plant RAD52 analogue has the nucleic acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO: 8 or fragment thereof.

24. The method of claim 21, said method comprises introducing into the plant cell an exogenous transcribable polynucleotide encoding an active fragment of a plant RAD52 analogue, wherein the fragment is lacking a mitochondria and/or chloroplast transit peptide.

25. The method of claim 19, said method comprises introducing into the plant cell a silencing molecule that silences the expression of said plant cell endogenous RAD52 analogue, thereby reducing the frequency of homologous recombination.

26. The method of claim 25, wherein the silencing molecule is selected from the group consisting of an RNA interference molecule, an antisense molecule and a ribozyme- encoding molecule.

27. The method of claim 26, wherein the silencing molecule is targeted to a polynucleotide having a nucleic acid sequence at least 50%> homologous to any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or a fragment thereof.

28. An isolated fragment of a plant RAD52 polypeptide capable of localizing a polynucleotide within a nucleus of a plant cell, the fragment having the amino acid sequence set forth in of SEQ ID NO:9.

29. The isolated fragment of claim 28, said fragment consists of SEQ ID NO:9.