A chromatin-associated protein required for inducing and limiting meiotic DNA double-strand break formation

Abstract

Programmed DNA double-strand breaks (DSBs) are required for meiotic recombination, but the number is strictly controlled because they are potentially harmful. Here we report a novel protein, Pars11, which is required for Spo11-dependent DSB formation in the protist Tetrahymena. Pars11 localizes to chromatin early in meiotic prophase in a Spo11-independent manner and is removed before the end of prophase. Pars11 removal depends on DSB formation and ATR-dependent phosphorylation. In the absence of the DNA damage sensor kinase ATR, Pars11 is retained on chromatin and excess DSBs are generated. Similar levels of Pars11 persistence and DSB overproduction occur in a non-phosphorylatable pars11 mutant. We conclude that Pars11 supports DSB formation by Spo11 until enough DSBs are formed; thereafter, DSB production stops in response to ATR-dependent degradation of Pars11 or its removal from chromatin. A similar DSB control mechanism involving a Rec114-Tel1/ATM-dependent negative feedback loop regulates DSB formation in budding yeast. However, there is no detectable sequence homology between Pars11 and Rec114, and DSB numbers are more tightly controlled by Pars11 than by Rec114. The discovery of this mechanism for DSB regulation in the evolutionarily distant protist and fungal lineages suggests that it is conserved across eukaryotes.

Spatial constraints on chromosomes are instrumental to meiotic pairing

“…As impaired nuclear elongation is characteristic of defective DSB formation (such as in the absence of SPO11 or PARS11 ) or DSB sensing (such as in the absence of ATR1 ) ( Loidl and Mochizuki, 2009 ; Mochizuki et al, 2008 ; Tian and Loidl, 2018 ; Fig. 2 ), DSB formation and processing was tested in the melg mutants.…”

“…Nuclear elongation is proposed to help the pairing of homologous chromosomes by arranging all chromosomes into a bundle of parallel strands in which corresponding regions become juxtaposed ( Loidl and Scherthan, 2004 ; Mochizuki et al, 2008 ). Nuclear reorganization is disturbed if DSBs are not formed, as in the spo11 Δ ( Mochizuki et al, 2008 ) or pars11 Δ mutants ( Tian and Loidl, 2018 ), DSBs are not sensed (upon suppression of the sensor kinase ATR) ( Loidl and Mochizuki, 2009 ) or MT polymerization is inhibited ( Kaczanowski et al, 1985 ; Loidl and Mochizuki, 2009 ). Under these conditions, either COs are not initiated or meiosis is arrested, which precludes an assessment of the role of nuclear reorganization in meiosis.…”

“…The polarized orientation of chromosome arms resembles the bouquet. Nuclear elongation depends on Spo11-induced DNA double-strand breaks (DSBs) ( Mochizuki et al, 2008 ; Tian and Loidl, 2018 ) and DSB sensing by the Atr1 kinase ( Loidl and Mochizuki, 2009 ), and is driven by intranuclear microtubules (MTs) ( Davidson and LaFountain, 1975 ; Kaczanowski et al, 1985 ; Loidl and Mochizuki, 2009 ; Wolfe et al, 1976 ).

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Non-coding RNA Transcription in Tetrahymena Meiotic Nuclei Requires Dedicated Mediator Complex-Associated Proteins

“…Total RNA was extracted from 5 mL samples of conjugating WT or mutant cells ($3 [70], PARS11 [67], TPB6, IES737 and IES4092 are listed in Data S2. Some of them are the same as previously described [36,67]. To examine scnRNA production, 10 mg total RNA was separated by 12% polyacrylamide-urea gel electrophoresis, as previously described [25].…”

“…Before the transformation, plasmid constructs for knocking out EMIT1 (using the neo4 cassette), EMIT2 (using the neo4 or chx cassette), and RIB1 (using the chx cassette) were linearized by NotI digestion. The linearized plasmid constructs or the DNA fragment assembled via overlapping PCR were introduced into starved B2086 and CU428 cells via biolistic transformation, and transformants were selected with increasing paromomycin or cycloheximide (Sigma-Aldrich, St Louis, MO, USA) concentration [67] until all MAC loci were replaced via phenotypic assortment [68]. Somatic gene knockout was confirmed by RT-PCR ( Figure S3D).…”