TetrahymenaMeiotic Nuclear Reorganization Is Induced by a Checkpoint Kinase–dependent Response to DNA Damage

Abstract

In the ciliate Tetrahymena, meiotic micronuclei (MICs) undergo extreme elongation, and meiotic pairing and recombination take place within these elongated nuclei (the "crescents"). We have previously shown that elongation does not occur in the absence of Spo11p-induced DNA double-strand breaks (DSBs). Here we show that elongation is restored in spo11⌬ mutants by various DNA-damaging agents including ones that may not cause DSBs to a notable extent. MIC elongation following Spo11p-induced DSBs or artificially induced DNA lesions is probably a DNA-damage response mediated by a phosphokinase signal transduction pathway, since it is suppressed by the ATM/ATR kinase inhibitors caffeine and wortmannin and by knocking out Tetrahymena's ATR orthologue. MIC elongation occurs concomitantly with the movement of centromeres away from the telomeric pole of the MIC. This DNA damage-dependent reorganization of the MIC helps to arrange homologous chromosomes alongside each other but is not sufficient for exact pairing. Thus, Spo11p contributes to bivalent formation in two ways: by creating a favorable spatial disposition of homologues and by stabilizing pairing by crossovers. The polarized chromosome orientation inside the crescent resembles the conserved meiotic bouquet, and crescent and bouquet also share the putative function of aiding meiotic pairing. However, they are regulated differently because in Tetrahymena, DSBs are required for entering rather than exiting this stage.

Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

“…This suggests that Spo11-DSBs form in an environment that is refractory to Rad53 recruitment and modification, but that there also are environments where exogenouslyinduced damage can trigger the mitotic DNA damage response. In light of this suggestion, it is interesting that the meiotic defects of spo11 mutants in a variety of organisms are often only partially rescued by DSBs caused by exogenous agents (Bowring et al, 2006;Celerin et al, 2000;Dernburg et al, 1998;Loidl and Mochizuki, 2009;Pauklin et al, 2009;Storlazzi et al, 2003;Thorne and Byers, 1993). While other factors may be responsible for the limited rescue observed, we suggest that it reflects the random location of exogenously-induced DSBs, with only a subset Figure 6.…”

The Tetrahymena meiotic chromosome bouquet is organized by centromeres and promotes interhomolog recombination

“…When nocodazole was added 110 min after meiosis induction, elongation was completely suppressed (no elongated nuclei in 200 conjugating cells scored 3.5 h after meiosis induction, as compared to 59% in the wild type at the same stage). Moreover, centromeres failed to cluster (Loidl and Mochizuki, 2009) (Fig. 3A).…”

“…The meiotic nuclei will first become drop-shaped, then spindle-shaped, before elongation culminates in the so-called crescent. During elongation, DSBs are formed, recombination protein Dmc1 forms foci on chromatin, and homologous chromosomes become juxtaposed (Loidl and Mochizuki, 2009;Lukaszewicz et al, 2010;Howard-Till et al, 2011). Later, nuclei shorten and chromatin condenses into threads.…”

“…S1E). At the beginning of nuclear elongation, centromeres become transiently dispersed and re-assemble in the fully elongated nucleus (Mochizuki et al, 2008;Loidl and Mochizuki, 2009). As expected, nocodazole efficiently prevented nuclear elongation.…”

“…However, nuclear elongation and centromere clustering are restored by artificially induced DNA lesions (Mochizuki et al, 2008). The bouquet is also dependent on the DNA damage sensor kinase ATR (Loidl and Mochizuki, 2009). Hence, it was hypothesized that nuclear elongation is a DNA damage response that helps or is required to repair DSBs.…”