engleski

MRE11 in genome stability in plants

To better understand the role of the MRE11 gene in Arabidopsis, we performed a comparative analysis of the several mre11 alleles, which harbored T-DNA insertion in different parts of the gene. A new allele mre11-4 has been molecularly characterized and compared with previously characterized alleles mre11-2 and mre11-3. Mutants of the ATM gene were analyzed in parallel. The mre11-4 and mre11-2 alleles presumably produce truncated MRE11 proteins composed of the first 499 and 529 amino acids, respectively. Although the putative MRE11 truncated proteins differ only by 30 amino acids, the mutants exhibited strikingly different phenotypes in regards to growth morphology, genome stability and meiosis. While the mre11-2 mutants are fully fertile and undergo normal meiosis, the mre11-4 plants are sterile due to aberrant repair of meiotic DNA breaks. Structural homology analysis suggests that the T-DNA insertion in the mre11-4 allele probably disrupted the putative RAD50 interaction and/or homodimerization domain, which is assumed to be preserved in mre11-2 allele. Intriguingly, introgression of the atm- 2 allele into the mre11-2 background renders the double mutant infertile, a phenotype not observed in either parent line. This data indicate that MRE11 partially compensates for ATM deficiency in meiosis of Arabidopsis. To investigate the role of MRE11 in the DNA damage response (DDR) and DNA repair, we have undertaken a new cytogenetic approach. Inflorescences of Arabidopsis were cut off and treated with bleomycin – a radiomimetic drug. The mitotic activity and chromosomal abnormalities were analyzed from pistil cells after 24, 48 and 72 hours of recovery periods. Wild – type (wt) plants showed reduced mitotic activity coupled with extensive chromosome abnormalities after 24 hours of recovery, but mitotic activity reached nearly normal level after the 72 hours of recovery period, with also lower rate of chromosomal abnormalities. All three mre11 mutants initially had slightly lower mitotic indices than wt, but after bleomycin treatment they reduced mitotic activities only slightly, in spite of extensive chromosome fusions and fragmentation which gradually deteriorated. The atm mutant line mirrored the behavior of mre11 mutants. These results suggest that mre11 and atm mutants failed to arrest cell cycle upon bleomycin treatment, but also that the DNA lesions were quickly and mostly erroneously repaired. These conclusions have been additionally confirmed by the results of RT-PCR which showed that bleomycin-treated mre11 and atm mutants were unable to activate a transcriptional response of the selected genes involved in DDR and DNA repair. The strong transcriptional downregulation of BRCA1, RAD51 and PARP1 genes in mre11 and atm mutants after bleomycin treatment was further demonstrated by real-time PCR. To correlate an increased level of DNA damage in mre11 mutants with the role of MRE11 in homologous recombination (HR) related mechanisms, we measured the frequency of intrachromosomal recombination events using an in planta GUS recombination reporter. mre11 mutants showed remarkably reduced number of recombination events per plant as compared to wt, either spontaneous or bleomycin induced. Taken together these data indicate the use of non-homologous end joining pathways instead of HR in MRE11 deficient plants.

MRE11; Arabidopsis thaliana; DNA damage response; genome instability; somatic homologous recombination; DNA repair genes transcription response

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