PT Unknown
AU Villar Fernandez, M
TI Biochemical and functional characterization of the Pch2/ORC AAA+ assembly in controlling meiotic DNA break formation
PD 07
PY 2020
DI 10.17185/duepublico/72234
LA en
AB Programmed DNA double-strand break (DSB) formation followed by homologous crossover recombination is crucial to establish physical linkages between homologous chromosomes and to ensure faithful chromosome segregation during meiosis. However, the introduction of DSBs within repetitive DNA elements is a major source of genomic instability, due to the possibility of DSBs repairing by non-allelic homologous recombination (NAHR). Indeed, DSBs in such repetitive regions can cause genome rearrangements and chromosome missegregation, which is linked to several genetic disorders and birth defects in humans.

	In this study, we investigated how the repetitive ribosomal DNA (rDNA)-boundaries of the budding yeast Saccharomyces cerevisiae, which are also at high risk of NAHR, are protected against the introduction of DSBs. Particularly, we focused on the interplay of two AAA+ ATPases, the meiosis-specific Pch2 and the Orc1 subunit of the hetero-hexameric Origin Recognition Complex (ORC; Orc1-Orc6), which are involved in the protection of the rDNA edges against DSBs. We demonstrated that in vivo Pch2 associates not only with the Orc1 subunit but also with Orc2 and Orc5, suggesting that Pch2 is able to associate with the entire ORC during meiotic G2/prophase. In addition, we reconstituted this macromolecular Pch2-ORC assembly and showed, by cross-link mass-spectrometry (XL-MS), that both the NH2-terminal domain (NTD) and the AAA+ domain of Pch2 are involved in the interaction with ORC. Moreover, we showed that deletion of the NTD of Pch2 severely impairs the interaction with ORC in vivo and in vitro and that the Pch2-AAA+ domain alone is not able to prevent rDNA-associated DSB formation. In addition, we delineated the minimal region within the NTD of Pch2 (amino acids 2-144) that is sufficient to recapitulate binding with ORC in vitro. We also identified residues within this region that are critical for establishing an association with ORC and for Pch2-Orc1 functionality at the rDNA. 

	To understand the role of other ORC subunits in the rDNA protection against meiotic DSB formation, we developed experimental approaches to functionally deplete selected ORC members (Orc2 and Orc5). Using this system, we revealed that Pch2-Orc1 functionality at the rDNA can occur in conditions of nuclear depletion of these subunits. By performing chromatin immunoprecipitation coupled to quantitative PCR (ChIP-qPCR) we showed that the function of Pch2-Orc1 at the rDNA does not require binding of ORC to its canonical sites (i.e. origins of replication). Furthermore, we have provided evidence that a chromatin-binding module of Orc1 (the bromo-adjacent homology (BAH) domain) is necessary for Pch2-ORC binding in vivo, besides not been directly involved in the in vitro binding of these complexes. These data suggests that in vivo interaction between both AAA+ ATPases is influenced by the local chromatin environment, which might be a reflect of the ability of Orc1-BAH domain to associate with regions with specific nucleosome features and in turn, to direct Orc1/ORC to sites distinct from origins of replication, enabling recruitment of Pch2 to these sites.

	Altogether, the data presented in this study demonstrate a direct interaction between Pch2 and ORC and reveals that, within ORC, Orc1 might have a critical contribution to both binding and functionality of this meiotic anti-DSB system. The fact that the function of Pch2-Orc1 in locally suppressing DSBs during meiotic G2/prophase presumably does not depend on the association of ORC to origins of replication likely represents an origin-independent role of Orc1/ORC. This role is distinct from its canonical one in DNA replication and seems to indicate that during meiotic G2/prophase, Orc1/ORC is repurposed to perform a function with the meiosis-specific AAA+ ATPase Pch2.
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