Biochemical and functional characterization of a meiosis-specific Pch2/ORC AAA+ assembly.

María Ascensión Villar-Fernández,Richard Cardoso Da Silva,John R Weir,Tanja Bange,Magdalena Firlej,Gerben Vader,Vivek B Raina,Annika Sarembe,Elisabeth Weir,Dongqing Pan

doi:10.26508/lsa.201900630

María Ascensión Villar-Fernández, Richard Cardoso Da Silva + Show 8 more

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https://doi.org/10.26508/lsa.201900630

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Abstract

Pch2 is a meiosis-specific AAA+ protein that controls several important chromosomal processes. We previously demonstrated that Orc1, a subunit of the ORC, functionally interacts with budding yeast Pch2. The ORC (Orc1-6) AAA+ complex loads the AAA+ MCM helicase to origins of replication, but whether and how ORC collaborates with Pch2 remains unclear. Here, we show that a Pch2 hexamer directly associates with ORC during the meiotic G2/prophase. Biochemical analysis suggests that Pch2 uses its non-enzymatic NH2-terminal domain and AAA+ core and likely engages the interface of ORC that also binds to Cdc6, a factor crucial for ORC-MCM binding. Canonical ORC function requires association with origins, but we show here that despite causing efficient removal of Orc1 from origins, nuclear depletion of Orc2 and Orc5 does not trigger Pch2/Orc1-like meiotic phenotypes. This suggests that the function for Orc1/Pch2 in meiosis can be executed without efficient association of ORC with origins of replication. In conclusion, we uncover distinct functionalities for Orc1/ORC that drive the establishment of a non-canonical, meiosis-specific AAA+ assembly with Pch2.

Highlights

Meiosis is a specialized cell division program that produces haploid gametes that are required for sexual reproduction
Considering that mutations in the Walker A motif lead to monomerization of Pch2 in vivo (Herruzo et al, 2016), our data suggest that the efficient interaction between Pch2 and Orc1 relies on ATP binding and Pch2 hexamer formation
Pch2 is an important regulator of meiosis, and it uses its enzymatic activity to influence the chromosomal association of its clients

Summary

Introduction

Meiosis is a specialized cell division program that produces haploid gametes that are required for sexual reproduction. Ploidy reduction requires several meiosis-specific events, which occur in the context of a highly orchestrated meiotic program (Petronczki et al, 2003). DSB formation and recombination are essential for meiosis, but errors that occur during these events endanger genome stability of the developing gametes (Sasaki et al, 2010). Pch forms homo-hexamers and uses its enzymatic activity to remodel (and affect the function of clients) (Chen et al, 2014; Ye et al, 2015; Ye et al, 2017; Alfieri et al, 2018). HORMA domain–containing proteins are confirmed Pch clients, and many, if not all, functions ascribed to Pch can be explained by its enzymatic activity toward HORMA proteins (Vader, 2015)

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