scholarly journals A meiosis-specific AAA+ assembly reveals repurposing of ORC during budding yeast gametogenesis

2019 ◽  
Author(s):  
María Ascensión Villar-Fernández ◽  
Richard Cardoso da Silva ◽  
Dongqing Pan ◽  
Elisabeth Weir ◽  
Annika Sarembe ◽  
...  
Keyword(s):  
Biochemical Analysis ◽  
Budding Yeast ◽  
Replication Origins ◽  
Terminal Domain ◽  
Binding Interface ◽  
Mcm Helicase ◽  
A Subunit ◽  
Per Se ◽  
Aaa Protein

ABSTRACTORC (Orc1-6) is an AAA+ complex that loads the AAA+ MCM helicase to replication origins. Orc1, a subunit of ORC, functionally interacts with budding yeast Pch2, a meiosis-specific AAA+ protein. Pch2 regulates several chromosomal events of gametogenesis, but mechanisms that dictate Pch2 function remain poorly understood. We demonstrate that ORC directly interacts with an AAA+ Pch2 hexamer. The ORC-Pch2 assembly is established without Cdc6, a factor crucial for ORC-MCM binding. Biochemical analysis suggests that Pch2 utilizes ORC’s Cdc6-binding interface and employs its non-enzymatic NH2-terminal domain and AAA+ core to engage ORC. In contrast to phenotypes observed upon Orc1 impairment, nuclear depletion of other subunits of ORC does not lead to Pch2-like phenotypes, indicating that ORC integrity per se is not required to support Pch2 function. We thus reveal functional interplay between Pch2 and ORC, and uncover the repurposing of ORC to establish a non-canonical and meiosis-specific AAA+ assembly.

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

2020 ◽  
Vol 3 (11) ◽  
pp. e201900630
Author(s):  
María Ascensión Villar-Fernández ◽  
Richard Cardoso da Silva ◽  
Magdalena Firlej ◽  
Dongqing Pan ◽  
Elisabeth Weir ◽  
...  
Keyword(s):  
Biochemical Analysis ◽  
Budding Yeast ◽  
Functional Characterization ◽  
Efficient Removal ◽  
Terminal Domain ◽  
Origins Of Replication ◽  
Mcm Helicase ◽  
A Subunit ◽  
Aaa Protein

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.

scholarly journals The pivot point arginines identified in the β-pinwheel structure of C-terminal domain from Salmonella Typhi DNA Gyrase A subunit

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ekta Sachdeva ◽  
Gurpreet Kaur ◽  
Pragya Tiwari ◽  
Deepali Gupta ◽  
Tej P. Singh ◽  
...  
Keyword(s):  
Dna Gyrase ◽  
Salmonella Typhi ◽  
Pivot Point ◽  
Terminal Domain ◽  
Gyrase A ◽  
A Subunit

scholarly journals Structural and functional analyses of the N-terminal domain of the A subunit of aBacillus megateriumspore germinant receptor

2019 ◽  
Vol 116 (23) ◽  
pp. 11470-11479 ◽  
Author(s):  
Yunfeng Li ◽  
Kai Jin ◽  
Abigail Perez-Valdespino ◽  
Kyle Federkiewicz ◽  
Andrew Davis ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Abc Transporters ◽  
Spore Germination ◽  
Critical Role ◽  
Chemical Shift Perturbation ◽  
Terminal Domain ◽  
Proposed Model ◽  
Strong Homology ◽  
A Subunit ◽  
Functional Analyses

Germination ofBacillusspores is induced by the interaction of specific nutrient molecules with germinant receptors (GRs) localized in the spore’s inner membrane. GRs typically consist of three subunits referred to as A, B, and C, although functions of individual subunits are not known. Here we present the crystal structure of the N-terminal domain (NTD) of the A subunit of theBacillus megateriumGerK3GR, revealing two distinct globular subdomains bisected by a cleft, a fold with strong homology to substrate-binding proteins in bacterial ABC transporters. Molecular docking, chemical shift perturbation measurement, and mutagenesis coupled with spore germination analyses support a proposed model that the interface between the two subdomains in the NTD of GR A subunits serves as the germinant binding site and plays a critical role in spore germination. Our findings provide a conceptual framework for understanding the germinant recruitment mechanism by which GRs trigger spore germination.

scholarly journals Mcm7, a Subunit of the Presumptive MCM Helicase, Modulates Its Own Expression in Conjunction with Mcm1

2003 ◽  
Vol 278 (28) ◽  
pp. 25408-25416 ◽  
Author(s):  
Michael J. Fitch ◽  
Justin J. Donato ◽  
Bik K. Tye
Keyword(s):  
Mcm Helicase ◽  
A Subunit

scholarly journals The key DNA-binding residues in the C-terminal domain of Mycobacterium tuberculosis DNA gyrase A subunit (GyrA)

2006 ◽  
Vol 34 (19) ◽  
pp. 5650-5659 ◽  
Author(s):  
You-Yi Huang ◽  
Jiao-Yu Deng ◽  
Jing Gu ◽  
Zhi-Ping Zhang ◽  
Anthony Maxwell ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Binding ◽  
Dna Gyrase ◽  
Terminal Domain ◽  
Gyrase A ◽  
A Subunit ◽  
Binding Residues

scholarly journals Activation of Silent Replication Origins at Autonomously Replicating Sequence Elements near the HML Locus in Budding Yeast

1999 ◽  
Vol 19 (9) ◽  
pp. 6098-6109 ◽  
Author(s):  
Marija Vujcic ◽  
Charles A. Miller ◽  
David Kowalski
Keyword(s):  
Replication Origin ◽  
Budding Yeast ◽  
Transcriptional Silencing ◽  
Growth Conditions ◽  
Replication Origins ◽  
Additional Time ◽  
Autonomously Replicating Sequence ◽  
Ars Elements ◽  
Active Replication ◽  
Chromosome Iii

ABSTRACT In the budding yeast, Saccharomyces cerevisiae, replicators can function outside the chromosome as autonomously replicating sequence (ARS) elements; however, within chromosome III, certain ARSs near the transcriptionally silent HML locus show no replication origin activity. Two of these ARSs comprise the transcriptional silencers E (ARS301) and I (ARS302). Another, ARS303, resides betweenHML and the CHA1 gene, and its function is not known. Here we further localized and characterized ARS303and in the process discovered a new ARS, ARS320. BothARS303 and ARS320 are competent as chromosomal replication origins since origin activity was seen when they were inserted at a different position in chromosome III. However, at their native locations, where the two ARSs are in a cluster withARS302, the I silencer, no replication origin activity was detected regardless of yeast mating type, special growth conditions that induce the transcriptionally repressed CHA1 gene,trans-acting mutations that abrogate transcriptional silencing at HML (sir3, orc5), orcis-acting mutations that delete the E and I silencers containing ARS elements. These results suggest that, for theHML ARS cluster (ARS303, ARS320, and ARS302), inactivity of origins is independent of local transcriptional silencing, even though origins and silencers share keycis- and trans-acting components. Surprisingly, deletion of active replication origins located 25 kb (ORI305) and 59 kb (ORI306) away led to detection of replication origin function at theHML ARS cluster, as well as at ARS301, the E silencer. Thus, replication origin silencing at HML ARSs is mediated by active replication origins residing at long distances fromHML in the chromosome. The distal active origins are known to fire early in S phase, and we propose that their inactivation delays replication fork arrival at HML, providing additional time for HML ARSs to fire as origins.

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