title: The requirement for recombination factors differs considerably between different pathways of homologous double-strand break repair in somatic plant cells.
abstract: In recent years, multiple factors involved in DNA double-strand break (DSB) repair have been characterised in Arabidopsis thaliana. Using homologous sequences in somatic cells, DSBs are mainly repaired by two different pathways: synthesis-dependent strand annealing (SDSA) and single-strand annealing (SSA). By applying recombination substrates in which recombination is initiated by the induction of a site-specific DSB by the homing endonuclease I-SceI, we were able to characterise the involvement of different factors in both pathways. The nucleases MRE11 and COM1, both involved in DSB end processing, were not required for either SDSA or SSA in our assay system. Both SDSA and SSA were even more efficient without MRE11, in accordance with the fact that a loss of MRE11 might negatively affect the efficiency of non-homologous end joining. Loss of the classical recombinase RAD51 or its two paralogues RAD51C and XRCC3, as well as the SWI2/SNF2 remodelling factor RAD54, resulted in a drastic deficiency in SDSA but had hardly any influence on SSA, confirming that a strand exchange reaction is only required for SDSA. The helicase FANCM, which is postulated to be involved in the stabilisation of recombination intermediates, is surprisingly not only needed for SDSA but to a lesser extent also for SSA. Both SSA and SDSA were affected only weakly when the SMC6B protein, implicated in sister chromatid recombination, was absent, indicating that SSA and SDSA are in most cases intrachromatid recombination reactions.
authors.affiliation: Botany II, Karlsruhe Institute of Technology, Kaiserstrasse 12, D-76131 Karlsruhe, Germany.
authors.forename: Nadine
authors.initials: N
authors.lastname: Roth
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Klimesch
authors.forename: Stefanie
authors.initials: S
authors.lastname: Dukowic-Schulze
authors.forename: Michael
authors.initials: M
authors.lastname: Pacher
authors.forename: Anja
authors.initials: A
authors.lastname: Mannuss
authors.forename: Holger
authors.initials: H
authors.lastname: Puchta
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D059765:Homologous Recombination; D000076228:MRE11 Homologue Protein; D030821:Plants, Genetically Modified; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic
chemical_list: D029681:Arabidopsis Proteins; C464449:At5g57450 protein, Arabidopsis; C414986:AtGR1 protein, Arabidopsis; C464448:AtRAD51C protein, Arabidopsis; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C492900:Mre11 protein, Arabidopsis; C518800:RAD54 protein, Arabidopsis; C580851:SMC6B protein, Arabidopsis; C495146:ATRAD51 protein, Arabidopsis; D051135:Rad51 Recombinase; D000076228:MRE11 Homologue Protein; D004265:DNA Helicases; C573664:FANCM protein, Arabidopsis