Characterization of Pch2 localization determinants reveals a nucleolar-independent role in the meiotic recombination checkpoint

Abstract

The meiotic recombination checkpoint blocks meiotic cell cycle progression in response to synapsis and/or recombination defects to prevent aberrant chromosome segregation. The evolutionarily-conserved budding yeast Pch2 TRIP13 AAA+ ATPase participates in this pathway by supporting phosphorylation of the Hop1 HORMAD adaptor at T318. In the wild type, Pch2 localizes to synapsed chromosomes and to the unsynapsed rDNA region (nucleolus), excluding Hop1. In contrast, in synaptonemal complex (SC)-defective zip1Δ mutants, which undergo checkpoint activation, Pch2 is detected only on the nucleolus. Alterations in some epigenetic marks that lead to Pch2 dispersion from the nucleolus suppress zip1Δ-induced checkpoint arrest. These observations have led to the notion that Pch2 nucleolar localization could be important for the meiotic recombination checkpoint. Here we investigate how Pch2 chromosomal distribution impacts on checkpoint function. We have generated and characterized several mutations that alter Pch2 localization pattern resulting in aberrant Hop1 distribution and compromised meiotic checkpoint response. Besides the AAA+ signature, we have identified a basic motif in the extended N-terminal domain critical for Pch2's checkpoint function and localization. We have also examined the functional relevance of the described Orc1-Pch2 interaction. Both proteins colocalize in the rDNA, and Orc1 depletion during meiotic prophase prevents Pch2 targeting to the rDNA allowing unwanted Hop1 accumulation on this region. However, Pch2 association with SC components remains intact in the absence of Orc1. We finally show that checkpoint activation is not affected by the lack of Orc1 demonstrating that, in contrast to previous hypotheses, nucleolar localization of Pch2 is actually dispensable for the meiotic checkpoint.

Phospho-Regulation of Meiotic Prophase

“…A plausible model is that Pch2 TRIP13 -mediated accumulation of unbuckled Hop1 HORMAD in the nucleoplasm leads to a cell cycle delay whereas an accumulation of unbuckled Hop1 HORMAD on chromosomes leads to a checkpoint silencing (Raina and Vader, 2020). Consistent with this model Pch2 TRIP13 executes its checkpoint-activating function when not bound to chromosomes (Herruzo et al, 2019), and Pch2 TRIP13 -dependent action on chromosomal Hop1 HORMAD silences Mek1 CHK2dependent signaling (Subramanian et al, 2016). The proposed action of Pch2 TRIP13 on Hop1 HORMAD is reminiscent of the function of TRIP13 and MAD2 in the spindle checkpoint (Vader, 2015), and nicely explains why pch2 mutations tend to alleviate arrests caused by synapsis defects but often strengthen arrests caused by DNA repair defects (Raina and Vader, 2020).…”

Homeostatic Control of Meiotic Prophase Checkpoint Function by Pch2 and Hop1

“…SC polymerization is dependent on repair of DSBs, and the recruitment of Pch2 is contingent on SC polymerization [19][20][21][22][23][24][25]. The requirement for Pch2 (in combination with reduced Hop1 levels) in mediating checkpoint function observed in both dmc1D and zip1D [25] points to a non-chromosomal role for Pch2 in checkpoint activation [32] in dmc1D and zip1D cells. Thus, these data suggest that, similar to the contribution of Pch2/TRIP13 to SAC activation (i.e., by maintaining sufficient amounts of O-Mad2 to fuel kinetochore based-SAC signaling) [48][49][50][51][52], Pch2 is needed to maintain the levels of ''signalingcompetent'' Hop1 required for incorporation into ''checkpointactive'' chromosomal sites [69].…”

“…In a role for Pch2 in synapsis checkpoint function [25,31]. In zip1D cells, Pch2 is not associated with chromosomes, except with nucleolar chromatin, but this pool of Pch2 is unlikely to contribute to checkpoint function [25,32]. The requirement for Pch2 in checkpoint function in zip1D is seemingly at odds with the described antagonistic role of Pch2 toward Hop1/RHMcbased checkpoint signaling that is associated with Zip1-dependent chromosomal recruitment [7,23,26,27].…”

DOT-1.1-dependent H3K79 methylation promotes normal meiotic progression and meiotic checkpoint function in C. elegans

“…In the checkpoint-defective yeast zip1Δ dot1Δ mutant, Pch2 is not retained in the nucleolus and instead it distributes throughout chromatin leading to the proposal that regulation of Pch2 nucleolar localization by Dot1 is important for checkpoint function [69,70]. However, more recent studies have demonstrated that the Pch2 protein also localizes in the cytoplasm of yeast cells, and that the presence of Pch2 in the nucleolus is actually dispensable for checkpoint function [71]. In syp-1 worms, PCH-2 is not detected associated to chromatin [47] (Fig 7), but the synapsis checkpoint is active [45] (Fig 6A).…”