SummaryDifferent DNA repair pathways that use homologous sequences in close proximity to genomic doublestrand breaks (DSBs) result in either an internal deletion or a gene conversion. We determined the ef®ciency of these pathways in somatic plant cells of transgenic Arabidopsis lines by monitoring the restoration of the b-glucuronidase (GUS) marker gene. The transgenes contain a recognition site for the restriction endonuclease I-SceI either between direct GUS repeats to detect deletion formation (DGU.US), or within the GUS gene to detect gene conversion using a nearby donor sequence in direct or inverted orientation (DU.GUS and IU.GUS). Without expression of I-SceI, the frequency of homologous recombination (HR) was low and similar for all three constructs. By crossing the different lines with an I-SceI expressing line, DSB repair was induced, and resulted in one to two orders of magnitude higher recombination frequency. The frequencies obtained with the DGU.US construct were about ®ve times higher than those obtained with DU.GUS and IU.GUS, irrespective of the orientation of the donor sequence. Our results indicate that recombination associated with deletions is the most ef®cient pathway of homologous DSB repair in plants. However, DSB-induced gene conversion seems to be frequent enough to play a signi®cant role in the evolution of tandemly arranged gene families like resistance genes.
Because of the availability of the complete sequence of the genome of the model plant Arabidopsis and of insertion mutants for most genes in public mutant collections, the elucidation of the particular role of different factors involved in DNA recombination and repair processes, an important task for plant biology, is becoming feasible. An assay system based on transgenes harboring homologous overlaps of the beta-glucuronidase (uidA) gene is available to determine recombination behavior in various mutant backgrounds. Restoration of the marker gene by homologous recombination can be detected by histochemical staining in planta. Inclusion of a site of the rare cutting restriction enzyme I-SceI in the transgene construct enables the determination of recombination frequencies after induction of double-strand breaks. In this chapter we describe how the respective transgene is transferred by transformation or crossing into the mutant background, how recombination frequencies are determined, and, if necessary, how cells carrying a restored uidA gene can be isolated and propagated for molecular analysis of the particular recombination event.
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