RESUMO
DNA double-strand breaks (DSBs) pose one of the most severe threats to genome integrity, potentially leading to cell death. After detection of a DSB, the DNA damage and repair response is initiated and the DSB is repaired by non-homologous end joining and/or homologous recombination. Many components of these processes are still unknown in Arabidopsis thaliana. In this work, we characterized γ-irradiation and mitomycin C induced 1 (GMI1), a member of the SMC-hinge domain-containing protein family. RT-PCR analysis and promoter-GUS fusion studies showed that γ-irradiation, the radio-mimetic drug bleocin, and the DNA cross-linking agent mitomycin C strongly enhance GMI1 expression particularly in meristematic tissues. The induction of GMI1 by γ-irradiation depends on the signalling kinase Ataxia telangiectasia-mutated (ATM) but not on ATM and Rad3-related (ATR). Epistasis analysis of single and double mutants demonstrated that ATM acts upstream of GMI1 while the atr gmi1-2 double mutant was more sensitive than the respective single mutants. Comet assay revealed a reduced rate of DNA double-strand break repair in gmi1 mutants during the early recovery phase after exposure to bleocin. Moreover, the rate of homologous recombination of a reporter construct was strongly reduced in gmi1 mutant plants upon exposure to bleocin or mitomycin C. GMI1 is the first member of its protein family known to be involved in DNA repair.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cromossomos de Plantas/metabolismo , DNA de Plantas/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Clonagem Molecular , Ensaio Cometa , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Plantas/genética , Flores/efeitos dos fármacos , Flores/metabolismo , Flores/efeitos da radiação , Regulação da Expressão Gênica de Plantas , Fusão Gênica , Meristema/efeitos dos fármacos , Meristema/metabolismo , Meristema/efeitos da radiação , Análise em Microsséries , Mitomicina/farmacologia , Mutagênese Insercional , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Plântula/efeitos dos fármacos , Plântula/genética , Transcrição GênicaRESUMO
Meiotic cohesin serves in sister chromatid linkage and DNA repair until its subunit Rec8 is cleaved by separase. Separase is activated when its inhibitor, securin, is polyubiquitinated by the Cdc20 regulated anaphase-promoting complex (APC(Cdc20)) and consequently degraded. Differently regulated APCs (APC(Cdh1), APC(Ama1)) have not been implicated in securin degradation at meiosis I. We show that Mnd2, a factor known to associate with APC components, prevents premature securin degradation in meiosis by APC(Ama1). mnd2Delta cells lack linear chromosome axes and exhibit precocious sister chromatid separation, but deletion of AMA1 suppresses these defects. Besides securin, Sgo1, a protein essential for protection of centromeric cohesion during anaphase I, is also destabilized in mnd2delta cells. Mnd2's disappearance prior to anaphase II may activate APC(Ama1). Human oocytes may spend many years in meiotic prophase before maturation. Inhibitors of meiotic APC variants could prevent loss of chiasmata also in these cells, thereby guarding against aberrant chromosome segregation.
