Different pathways of homologous recombination are used for the repair of double-strand breaks within tandemly arranged sequences in the plant genome

Journal Article / 2003

Orel, Nadiya; Kyryk, Anzhela; Puchta, Holger

Different 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 ˜-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.