ATM controls meiotic DNA double-strand break formation and recombination and affects synaptonemal complex organization in plants

Abstract

Meiosis is a specialized cell division that gives rise to genetically distinct gametic cells. Meiosis relies on the tightly controlled formation of DNA double-strand breaks (DSBs) and their repair via homologous recombination for correct chromosome segregation. Like all forms of DNA damage, meiotic DSBs are potentially harmful and their formation activates an elaborate response to inhibit excessive DNA break formation and ensure successful repair. Previous studies established the protein kinase ATM as a DSB sensor and meiotic regulator in several organisms. Here we show that Arabidopsis ATM acts at multiple steps during DSB formation and processing, as well as crossover (CO) formation and synaptonemal complex (SC) organization, all vital for the successful completion of meiosis. We developed a single-molecule approach to quantify meiotic breaks and determined that ATM is essential to limit the number of meiotic DSBs. Local and genome-wide recombination screens showed that ATM restricts the number of interference-insensitive COs, while super-resolution STED nanoscopy of meiotic chromosomes revealed that the kinase affects chromatin loop size and SC length and width. Our study extends our understanding of how ATM functions during plant meiosis and establishes it as an integral factor of the meiotic program.

From Microscopy to Nanoscopy: Defining an Arabidopsis thaliana Meiotic Atlas at the Nanometer Scale

“…Each single focus found at an epifluorescence microscope is composed of several smaller foci at the STED (compare RAD51 staining in Figures 2, 5). While RAD51 foci, for example, were found to be mainly circular and peak in zygotene at around 200 foci per nucleus in widefield images (Kumar et al, 2019;Sims et al, 2019;Kurzbauer et al, 2021), more than 1,000 foci are found in images acquired by STED nanoscopy (Figure 5). In addition, foci seem to assume different shapes over time, with few larger clusters forming in pachytene and likely representing different repair intermediates.…”

“…These two stages are highly informative, because chromosome fragments are visible when DNA repair is defective (as in com1-1 , Figures 1G2 , H2 ) and chromosome missegregation can be observed when homolog interactions are reduced or absent (as in spo11-2-3 ; Figures 1G1 , H1 ; Grelon et al, 2001 ; Stacey et al, 2006 ). In addition, irregular repair might manifest in chromatin bridges, well visible during these (and subsequent) stages ( Figures 1H2 , L2 ; Uanschou et al, 2007 ; Kurzbauer et al, 2021 ) and cohesion-deficiencies may result in premature sister separation ( Cai et al, 2003 ; Chelysheva et al, 2005 ).…”

“…The FISH technique has been widely used within the meiotic plant community and is an essential tool to determine the chiasma frequency on individual chromosomes. To this end, bivalents are unequivocally identified by FISH labeling of the 5S and 45S rDNA regions and the number of chiasmata per chromosome arm is deduced from bivalent shape (Sanchez Moran et al, 2001;Lopez et al, 2012;Armstrong, 2013;Kurzbauer et al, 2018Kurzbauer et al, , 2021. Finally, single-molecule RNA-FISH has become increasingly popular to analyze transcription in plant tissues.…”

“…Synapsed chromosomes appear as thick, puffed up threads on acid spreads ( Figure 1C ) and staining for ZYP1 reveals full polymerization along chromosomes ( Figure 2 ). ZYP1 is therefore an ideal marker when measuring total SC length within a nucleus ( Drouaud et al, 2007 ; Lloyd et al, 2018 ; Kurzbauer et al, 2021 ). ASY1 can still be detected along pachytene chromosomes, but the staining is remarkably less bright apart from few brightly stained stretches ( Figure 2 ) that mark the nucleolus organizer regions (NORs) containing the 45S rDNA genes ( Fransz et al, 1998 ; Sims et al, 2019 ).…”

Phospho-Regulation of Meiotic Prophase

“…The number of meiotic DSBs per cell must be carefully titrated to ensure sufficient DSB formation for successful meiotic recombination while minimizing the risk of chromosome abnormalities associated with excessive DSB numbers. ATM mutants in various organisms exhibit increased DSB formation, suggesting a universal role for ATM in the regulation of DSB levels (Joyce et al, 2011;Lange et al, 2011;Checchi et al, 2014;Garcia et al, 2015;Mohibullah and Keeney, 2017;Fowler et al, 2018;Kurzbauer et al, 2021). In S. cerevisiae and S. pombe, ATM regulates DSB numbers by preventing double-cutting in proximity of DSBs (Garcia et al, 2015;Fowler et al, 2018).…”