title: A transient assay for recombination demonstrates that Arabidopsis SNM1 and XRCC3 enhance non-homologous recombination.
abstract: Replacement  of endogenous genes by homologous recombination is rare in plants; the majority  of genetic modifications are the result of transforming DNA molecules  undergoing random genomic insertion by way of non-homologous recombination.  Factors that affect chromatin remodeling and DNA repair are thought to have the  potential to enhance the frequency of homologous recombination in plants.  Conventional tools to study the frequencies of genetic recombination often rely  on stable transformation-based approaches, with these systems being rarely capable  of high-throughput or combinatorial analysis. We developed a series of vectors  that use chemiluminescent (LUC and REN) reporter genes to assay  the relative frequency of homologous and non-homologous recombination in  plants. These transient assay vectors were used to screen 14 candidate genes  for their effects on recombination frequencies in Nicotiana benthamiana plants.  Over-expression of Arabidopsis genes with sequence similarity to SNM1 from yeast and XRCC3 from humans enhanced the frequency of  non-homologous recombination when assayed using two different donor vectors.  Transient N. benthamiana leaf systems were also used in an alternative  assay for preliminary measurements of homologous recombination frequencies,  which were found to be enhanced by over-expression of RAD52, MIM and RAD51 from yeast, as well as CHR24 from Arabidopsis. The  findings for the assays described here are in line with previous studies that  analyzed recombination frequencies using stable transformation. The assays we  report have revealed functions in non-homologous recombination for the Arabidopsis  SNM1 and XRCC3 genes, so the suppression of these genes' expression  offers a potential means to enhance the gene targeting frequency in plants.  Furthermore, our findings also indicate that plant gene targeting frequencies  could be enhanced by over-expression of RAD52, MIM, CHR24,  and RAD51 genes.
authors.affiliation: The New Zealand Institute for Plant & Food Research Limited, Sandringham, Auckland, New Zealand. rjoh064@aucklanduni.ac.nz
authors.forename: R A
authors.initials: RA
authors.lastname: Johnson
authors.forename: R P
authors.initials: RP
authors.lastname: Hellens
authors.forename: D R
authors.initials: DR
authors.lastname: Love
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D042002:Chromatin Assembly and Disassembly; D004260:DNA Repair; D018744:DNA, Plant / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D017930:Genes, Reporter; D059765:Homologous Recombination / Q000235:genetics; D008840:Microfilament Proteins / Q000096:biosynthesis / Q000235:genetics; D051135:Rad51 Recombinase / Q000096:biosynthesis / Q000235:genetics; D051721:Rad52 DNA Repair and Recombination Protein / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D014026:Nicotiana / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; C464449:At5g57450 protein, Arabidopsis; D018744:DNA, Plant; D008840:Microfilament Proteins; D051721:Rad52 DNA Repair and Recombination Protein; C499103:SNM1 protein, Arabidopsis; C495146:ATRAD51 protein, Arabidopsis; D051135:Rad51 Recombinase