kcnj13 regulates pigment cell shapes in zebrafish and has diverged by cis-regulatory evolution between Danio species.

Teleost fish of the genus Danio are excellent models to study the genetic and cellular bases of pigment pattern variation in vertebrates. The two sister species Danio rerio and Danio aesculapii show divergent patterns of horizontal stripes and vertical bars that are partly caused by the divergence of the potassium channel gene kcnj13. Here, we show that kcnj13 is required only in melanophores for interactions with xanthophores and iridophores, which cause location-specific pigment cell shapes and thereby influence colour pattern and contrast in D. rerio. Cis-regulatory rather than protein coding changes underlie kcnj13 divergence between the two Danio species. Our results suggest that homotypic and heterotypic interactions between the pigment cells and their shapes diverged between species by quantitative changes in kcnj13 expression during pigment pattern diversification.

Reconstitution of a 26-Subunit Human Kinetochore Reveals Cooperative Microtubule Binding by CENP-OPQUR and NDC80.

The approximately thirty core subunits of kinetochores assemble on centromeric chromatin containing the histone H3 variant CENP-A and connect chromosomes with spindle microtubules. The chromatin proximal 16-subunit CCAN (constitutive centromere associated network) creates a mechanically stable bridge between CENP-A and the kinetochore's microtubule-binding machinery, the 10-subunit KMN assembly. Here, we reconstituted a stoichiometric 11-subunit human CCAN core that forms when the CENP-OPQUR complex binds to a joint interface on the CENP-HIKM and CENP-LN complexes. The resulting CCAN particle is globular and connects KMN and CENP-A in a 26-subunit recombinant particle. The disordered, basic N-terminal tail of CENP-Q binds microtubules and promotes accurate chromosome alignment, cooperating with KMN in microtubule binding. The N-terminal basic tail of the NDC80 complex, the microtubule-binding subunit of KMN, can functionally replace the CENP-Q tail. Our work dissects the connectivity and architecture of CCAN and reveals unexpected functional similarities between CENP-OPQUR and the NDC80 complex.

Biochemical characterisation of Mer3 helicase interactions and the protection of meiotic recombination intermediates.

Crossing over between homologs is critical for the stable segregation of chromosomes during the first meiotic division. Saccharomyces cerevisiae Mer3 (HFM1 in mammals) is a SF2 helicase and member of the ZMM group of proteins, that facilitates the formation of the majority of crossovers during meiosis. Here, we describe the structural organisation of Mer3 and using AlphaFold modelling and XL-MS we further characterise the previously described interaction with Mlh1-Mlh2. We find that Mer3 also forms a previously undescribed complex with the recombination regulating factors Top3 and Rmi1 and that this interaction is competitive with Sgs1BLM helicase. Using in vitro reconstituted D-loop assays we show that Mer3 inhibits the anti-recombination activity of Sgs1 helicase, but only in the presence of Dmc1. Thus we provide a mechanism whereby Mer3 interacts with a network of proteins to protect Dmc1 derived D-loops from dissolution.

Homologous chromosomes are stably conjoined for Drosophila male meiosis I by SUM, a multimerized protein assembly with modules for DNA-binding and for separase-mediated dissociation co-opted from cohesin.

For meiosis I, homologous chromosomes must be paired into bivalents. Maintenance of homolog conjunction in bivalents until anaphase I depends on crossovers in canonical meiosis. However, instead of crossovers, an alternative system achieves homolog conjunction during the achiasmate male meiosis of Drosophila melanogaster. The proteins SNM, UNO and MNM are likely constituents of a physical linkage that conjoins homologs in D. melanogaster spermatocytes. Here, we report that SNM binds tightly to the C-terminal region of UNO. This interaction is homologous to that of the cohesin subunits stromalin/Scc3/STAG and α-kleisin, as revealed by sequence similarities, structure modeling and cross-link mass spectrometry. Importantly, purified SU_C, the heterodimeric complex of SNM and the C-terminal region of UNO, displayed DNA-binding in vitro. DNA-binding was severely impaired by mutational elimination of positively charged residues from the C-terminal helix of UNO. Phenotypic analyses in flies fully confirmed the physiological relevance of this basic helix for chromosome-binding and homolog conjunction during male meiosis. Beyond DNA, SU_C also bound MNM, one of many isoforms expressed from the complex mod(mdg4) locus. This binding of MNM to SU_C was mediated by the MNM-specific C-terminal region, while the purified N-terminal part common to all Mod(mdg4) isoforms multimerized into hexamers in vitro. Similarly, the UNO N-terminal domain formed tetramers in vitro. Thus, we suggest that multimerization confers to SUM, the assemblies composed of SNM, UNO and MNM, the capacity to conjoin homologous chromosomes stably by the resultant multivalent DNA-binding. Moreover, to permit homolog separation during anaphase I, SUM is dissociated by separase, since UNO, the α-kleisin-related protein, includes a separase cleavage site. In support of this proposal, we demonstrate that UNO cleavage by tobacco etch virus protease is sufficient to release homolog conjunction in vivo after mutational exchange of the separase cleavage site with that of the bio-orthogonal protease.

The molecular machinery of meiotic recombination.

Meiotic recombination, a cornerstone of eukaryotic diversity and individual genetic identity, is essential for the creation of physical linkages between homologous chromosomes, facilitating their faithful segregation during meiosis I. This process requires that germ cells generate controlled DNA lesions within their own genome that are subsequently repaired in a specialised manner. Repair of these DNA breaks involves the modulation of existing homologous recombination repair pathways to generate crossovers between homologous chromosomes. Decades of genetic and cytological studies have identified a multitude of factors that are involved in meiotic recombination. Recent work has started to provide additional mechanistic insights into how these factors interact with one another, with DNA, and provide the molecular outcomes required for a successful meiosis. Here, we provide a review of the recent developments with a focus on protein structures and protein-protein interactions.

Fire management alters the thermal landscape and provides multi-scale thermal options for a terrestrial turtle facing a changing climate.

As effects of climate change intensify, there is a growing need to understand the thermal properties of landscapes and their influence on wildlife. A key thermal property of landscapes is vegetation structure and composition. Management approaches can alter vegetation and consequently the thermal landscape, potentially resulting in underappreciated consequences for wildlife thermoregulation. Consideration of spatial scale can clarify how management overlaid onto existing vegetation patterns affects thermal properties of landscapes relevant to wildlife. We examined effects of temperature, fire management, and vegetation structure on multi-scale habitat selection of an ectothermic vertebrate (the turtle Terrapene carolina triunguis) in the Great Plains of the central United States by linking time-since-fire data from 18 experimental burn plots to turtle telemetry locations and thermal and vegetation height data. Within three 60-ha experimental landscapes, each containing six 10-ha sub-blocks that are periodically burned, we found that turtles select time-since-fire gradients differently depending on maximum daily ambient temperature. At moderate temperatures, turtles selected sub-blocks with recent (<1 year) time-since-fire, but during relatively hot and cool conditions, they selected sub-blocks with later (2-3 year) time-since-fire that provided thermal buffering compared with recently burned sub-blocks. Within 10-ha sub-blocks, turtles selected locations with taller vegetation during warmer conditions that provided thermal buffering. Thermal performance curves revealed that turtle activity declined as temperatures exceeded ~24-29°C, and on "heat days" (≥29°C) 73% of turtles were inactive compared with 37% on non-heat days, emphasizing that thermal extremes may lead to opportunity costs (i.e., foregone benefits turtles could otherwise accrue if active). Our results indicate that management approaches that promote a mosaic of vegetation heights, like spatiotemporally dynamic fire, can provide thermal refuges at multiple spatial scales and thus be an actionable way to provide wildlife with multiple thermal options in the context of ongoing and future climate change.

Factors influencing the persistence of a fire-sensitive Artemisia species in a fire-dependent ecosystem.

Fire refugia and patchiness are important to the persistence of fire-sensitive species and may facilitate biodiversity conservation in fire-dependent landscapes. Playing the role of ecosystem engineers, large herbivores alter vegetation structure and can reduce wildfire risk. However, herbivore effects on the spatial variability of fire and the persistence of fire-sensitive species are not clear. To examine the hypothesis that large herbivores support the persistence of fire-sensitive species through the creation of fire refugia in fire-prone landscapes, we examined the response of a fire-sensitive plant, Wyoming big sagebrush (Artemisia tridentata ssp. wyomingensis [Beetle & Young]) to fire and grazing in the fire-dependent mixed-grass prairie of the northern Great Plains. We carried out a controlled burn in 2010 within pre-established exclosures that allowed differential access to wild and domestic herbivores and no record of fire in the previous 75 years due to fire suppression efforts. The experiment was set up with a split-plot design to also examine potential changes in plots that were not burned. Canopy cover of big sagebrush was recorded before the burn in 2010 and again in 2011 with percent area burned recorded within 1-month post-fire in the burned plots. Percentage area burned was the greatest in ungulate exclosures (92% ± 2%) and the least in open areas (55% ± 21%), suggesting that large herbivores influenced fire behavior (e.g., reducing fire intensity and rate of spread) and are likely to increase fire patchiness through their alterations to the fuel bed. Regression analysis indicated that the proportion of sagebrush cover lost was significantly correlated with the proportion of area burned (R2  = 0.76, p = 0.05). No differences in the non-burn plots were observed among grazing treatments or among years. Altogether, this illustrates the potential importance of large herbivores in creating biotic-driven fire refugia for fire-sensitive species to survive within the flammable fuel matrix of fire-dependent grassland ecosystems such as the mixed-grass prairie. Our findings also attest to the resiliency of the northern Great Plains to fire and herbivory and underscore the value of managing grasslands for heterogeneity with spatial and temporal variations in these historic disturbances.

Insights from biochemical reconstitution into the architecture of human kinetochores.

Chromosomes are carriers of genetic material and their accurate transfer from a mother cell to its two daughters during cell division is of paramount importance for life. Kinetochores are crucial for this process, as they connect chromosomes with microtubules in the mitotic spindle. Kinetochores are multi-subunit complexes that assemble on specialized chromatin domains, the centromeres, that are able to enrich nucleosomes containing the histone H3 variant centromeric protein A (CENP-A). A group of several additional CENPs, collectively known as constitutive centromere associated network (CCAN), establish the inner kinetochore, whereas a ten-subunit assembly known as the KMN network creates a microtubule-binding site in the outer kinetochore. Interactions between CENP-A and two CCAN subunits, CENP-C and CENP-N, have been previously described, but a comprehensive understanding of CCAN organization and of how it contributes to the selective recognition of CENP-A has been missing. Here we use biochemical reconstitution to unveil fundamental principles of kinetochore organization and function. We show that cooperative interactions of a seven-subunit CCAN subcomplex, the CHIKMLN complex, determine binding selectivity for CENP-A over H3-nucleosomes. The CENP-A:CHIKMLN complex binds directly to the KMN network, resulting in a 21-subunit complex that forms a minimal high-affinity linkage between CENP-A nucleosomes and microtubules in vitro. This structural module is related to fungal point kinetochores, which bind a single microtubule. Its convolution with multiple CENP-A proteins may give rise to the regional kinetochores of higher eukaryotes, which bind multiple microtubules. Biochemical reconstitution paves the way for mechanistic and quantitative analyses of kinetochores.

The molecular architecture of the TRAMP complex reveals the organization and interplay of its two catalytic activities.

The TRAMP complex is involved in the nuclear surveillance and turnover of noncoding RNAs and intergenic transcripts. TRAMP is associated with the nuclear exosome and consists of a poly(A)polymerase subcomplex (Trf4-Air2) and a helicase (Mtr4). We found that N-terminal low-complexity regions of Trf4 and Air2 bind Mtr4 in a cooperative manner. The 2.4 Å resolution crystal structure of the corresponding ternary complex reveals how Trf4 and Air2 wrap around the DExH core of the helicase. Structure-based mutations on the DExH core impair binding to Trf4 and Air2, and also to Trf5 and Air1. The combination of structural, biochemical, and biophysical data suggests that the poly(A)polymerase core of Trf4-Air2 is positioned below the base of the helicase, where the unwound 3' end of an RNA substrate is expected to emerge. The results reveal conceptual similarities between the two major regulators of the exosome, the nuclear TRAMP and cytoplasmic Ski complexes.

Determinants of perceived risk and liability concerns associated with prescribed burning in the United States.

While prescribed burning is a proven tool in the management of forests and grasslands, its use has been limited due, in part, to potential risks that may result in legal liability, property damage, and personal injury. The purpose of this study is to understand the factors that shape landowners' and fire professionals' perceptions of risks associated with prescribed burning activities. The data for this study were collected from active prescribed fire professionals involved in Prescribed Burn Association (PBA) activities in 14 Southern and Mid-western states. Perceived risk was higher among respondents with higher levels of concern related to safety and weather but lower among respondents with more experience in burning activities. Sociodemographic variables such as age and income were not significantly correlated with risk perception. These findings are useful for better understanding how landowners and fire professionals perceive risk and offer insight into how perceived risk affects decisions to apply prescribed burns.

Seeding the meiotic DNA break machinery and initiating recombination on chromosome axes.

Programmed DNA double-strand break (DSB) formation is a crucial feature of meiosis in most organisms. DSBs initiate recombination-mediated linking of homologous chromosomes, which enables correct chromosome segregation in meiosis. DSBs are generated on chromosome axes by heterooligomeric focal clusters of DSB-factors. Whereas DNA-driven protein condensation is thought to assemble the DSB-machinery, its targeting to chromosome axes is poorly understood. We uncover in mice that efficient biogenesis of DSB-machinery clusters requires seeding by axial IHO1 platforms. Both IHO1 phosphorylation and formation of axial IHO1 platforms are diminished by chemical inhibition of DBF4-dependent kinase (DDK), suggesting that DDK contributes to the control of the axial DSB-machinery. Furthermore, we show that axial IHO1 platforms are based on an interaction between IHO1 and the chromosomal axis component HORMAD1. IHO1-HORMAD1-mediated seeding of the DSB-machinery on axes ensures sufficiency of DSBs for efficient pairing of homologous chromosomes. Without IHO1-HORMAD1 interaction, residual DSBs depend on ANKRD31, which enhances both the seeding and the growth of DSB-machinery clusters. Thus, recombination initiation is ensured by complementary pathways that differentially support seeding and growth of DSB-machinery clusters, thereby synergistically enabling DSB-machinery condensation on chromosomal axes.

GTPase activation of elongation factor EF-Tu by the ribosome during decoding.

We have used single-particle reconstruction in cryo-electron microscopy to determine a structure of the Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF-Tu), tRNA and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin. This represents the state in the decoding process just after codon recognition by tRNA and the resulting GTP hydrolysis by EF-Tu, but before the release of EF-Tu from the ribosome. Progress in sample purification and image processing made it possible to reach a resolution of 6.4 A. Secondary structure elements in tRNA, EF-Tu and the ribosome, and even GDP and kirromycin, could all be visualized directly. The structure reveals a complex conformational rearrangement of the tRNA in the A/T state and the interactions with the functionally important switch regions of EF-Tu crucial to GTP hydrolysis. Thus, the structure provides insights into the molecular mechanism of signalling codon recognition from the decoding centre of the 30S subunit to the GTPase centre of EF-Tu.

Structural analysis reveals the characteristic features of Mtr4, a DExH helicase involved in nuclear RNA processing and surveillance.

Mtr4 is a conserved RNA helicase that functions together with the nuclear exosome. It participates in the processing of structured RNAs, including the maturation of 5.8S ribosomal RNA (rRNA). It also interacts with the polyadenylating Trf4-Air2 heterodimer to form the so-called TRAMP (Trf4-Air2-Mtr4 Polyadenylation) complex. TRAMP is involved in exosome-mediated degradation of aberrant RNAs in nuclear surveillance pathways. We report the 2.9-A resolution crystal structure of Saccharomyces cerevisiae Mtr4 in complex with ADP and RNA. The structure shows a central ATPase core similar to that of other DExH helicases. Inserted in the DExH core is a region characteristic of Mtr4 orthologues that folds into an elongated stalk connected to a beta-barrel domain. This domain shows unexpected similarity to the KOW domain of L24, a ribosomal protein that binds 23S rRNA. We find that indeed the KOW domain of Mtr4 is able to bind in vitro transcribed tRNA(iMet), suggesting it might assist in presenting RNA substrates to the helicase core. The interaction of Mtr4 with Trf4-Air2 is mediated not by the stalk/KOW insertion but by the DExH core. We find that in the context of the TRAMP complex, the DExH core functions independently in vitro as an RNA helicase and a protein-binding platform. Mtr4 has thus evolved specific structural and surface features to perform its multiple functions.

Unwinding during stressful times: Mechanisms of helicases in meiotic recombination.

Successful meiosis I requires that homologous chromosomes be correctly linked before they are segregated. In most organisms this physical linkage is achieved through the generation of crossovers between the homologs. Meiotic recombination co-opts and modifies the canonical homologous recombination pathway to successfully generate crossovers One of the central components of this pathway are a number of conserved DNA helicases. Helicases couple nucleic acid binding to nucleotide hydrolysis and use this activity to modify DNA or protein-DNA substrates. During meiosis I it is necessary for the cell to modulate the canonical DNA repair pathways in order to facilitate the generation of interhomolog crossovers. Many of these meiotic modulations take place in pathways involving DNA helicases, or with a meiosis specific helicase. This short review explores what is currently understood about these helicases, their interaction partners, and the role of regulatory modifications during meiosis I. We focus in particular on the molecular structure and mechanisms of these helicases.

Seeding the meiotic DNA break machinery and initiating recombination on chromosome axes.

Programmed DNA double-strand break (DSB) formation is a unique meiotic feature that initiates recombination-mediated linking of homologous chromosomes, thereby enabling chromosome number halving in meiosis. DSBs are generated on chromosome axes by heterooligomeric focal clusters of DSB-factors. Whereas DNA-driven protein condensation is thought to assemble the DSB-machinery, its targeting to chromosome axes is poorly understood. We discovered in mice that efficient biogenesis of DSB-machinery clusters requires seeding by axial IHO1 platforms, which are based on a DBF4-dependent kinase (DDK)-modulated interaction between IHO1 and the chromosomal axis component HORMAD1. IHO1-HORMAD1-mediated seeding of the DSB-machinery on axes ensures sufficiency of DSBs for efficient pairing of homologous chromosomes. Without IHO1-HORMAD1 interaction, residual DSBs depend on ANKRD31, which enhances both the seeding and the growth of DSB-machinery clusters. Thus, recombination initiation is ensured by complementary pathways that differentially support seeding and growth of DSB-machinery clusters, thereby synergistically enabling DSB-machinery condensation on chromosomal axes.

Silent recognition of flagellins from human gut commensal bacteria by Toll-like receptor 5.

Flagellin, the protein subunit of the bacterial flagellum, stimulates the innate immune receptor Toll-like receptor 5 (TLR5) after pattern recognition or evades TLR5 through lack of recognition. This binary response fails to explain the weak agonism of flagellins from commensal bacteria, raising the question of how TLR5 response is tuned. Here, we screened abundant flagellins present in metagenomes from human gut for both TLR5 recognition and activation and uncovered a class of flagellin-TLR5 interaction termed silent recognition. Silent flagellins were weak TLR5 agonists despite pattern recognition. Receptor activity was tuned by a TLR5-flagellin interaction distal to the site of pattern recognition that was present in Salmonella flagellin but absent in silent flagellins. This interaction enabled flagellin binding to preformed TLR5 dimers and increased TLR5 signaling by several orders of magnitude. Silent recognition by TLR5 occurred in human organoids and mice, and silent flagellin proteins were present in human stool. These flagellins were produced primarily by the abundant gut bacteria Lachnospiraceae and were enriched in nonindustrialized populations. Our findings provide a mechanism for the innate immune system to tolerate commensal-derived flagellins while remaining vigilant to the presence of flagellins produced by pathogens.

Tale of a twist: magnetic and optical switching in copper(II) semiquinone complexes.

An intermediate (C) that is observed in both phenol hydroxylation and catechol oxidation with the side-on peroxide species [Cu(2)O(2)(DBED)(2)](2+) (DBED = N(1),N(2)-di-tert-butylethane-1,2-diamine) is identified as a copper(II) semiquinone species ([1](+)) through independent synthesis and characterization. The reaction of the redox-active 3,5-di-tert-butylquinone ligand with [(DBED)Cu(I)(MeCN)](+) yields a copper(II) semiquinone [1](+) complex with a singlet ground state and an intense purple chromophore (ε(580) ~ 3500 M(-1) cm(-1)). All other copper(II) semiquinone complexes characterized to date are paramagnetic and weakly colored (ε(800) ~ 500 M(-1) cm(-1)). Antiferromagnetic coupling between the Cu(II) center and the semiquinone radical in [1](+) is characterized by paramagnetic (1)H NMR and SQUID magnetometry. Comparative X-ray crystal structures along with density functional theory calculations correlate the geometric structures of copper(II) semiquinone complexes with their magnetic and optical properties. The unique observable properties of [1](+) originate from an increase in the overlap of the Cu 3d and semiquinone π orbitals resulting from a large rhombic distortion in the structure with a twist of 51°, attributable to the large isotropic demands of the tert-butyl substituents of the DBED ligand. Independent characterization of [1](+) allows the spectroscopic yields of intermediate C to be quantified in this intriguing hydroxylation reaction.

InteBac: An integrated bacterial and baculovirus expression vector suite.

The successful production of recombinant protein for biochemical, biophysical, and structural biological studies critically depends on the correct expression organism. Currently, the most commonly used expression organisms for structural studies are Escherichia coli (~70% of all PDB structures) and the baculovirus/ insect cell expression system (~5% of all PDB structures). While insect cell expression is frequently successful for large eukaryotic proteins, it is relatively expensive and time-consuming compared to E. coli expression. Frequently the decision to carry out a baculovirus project means restarting cloning from scratch. Here we describe an integrated system that allows simultaneous cloning into E. coli and baculovirus expression vectors using the same PCR products. The system offers a flexible array of N- and C-terminal affinity, solubilization and utility tags, and the speed allows expression screening to be completed in E. coli, before carrying out time and cost-intensive experiments in baculovirus. Importantly, we describe a means of rapidly generating polycistronic bacterial constructs based on the hugely successful biGBac system, making InteBac of particular interest for researchers working on recombinant protein complexes.

Novel mechanistic insights into the role of Mer2 as the keystone of meiotic DNA break formation.

In meiosis, DNA double-strand break (DSB) formation by Spo11 initiates recombination and enables chromosome segregation. Numerous factors are required for Spo11 activity, and couple the DSB machinery to the development of a meiosis-specific 'axis-tethered loop' chromosome organisation. Through in vitro reconstitution and budding yeast genetics, we here provide architectural insight into the DSB machinery by focussing on a foundational DSB factor, Mer2. We characterise the interaction of Mer2 with the histone reader Spp1, and show that Mer2 directly associates with nucleosomes, likely highlighting a contribution of Mer2 to tethering DSB factors to chromatin. We reveal the biochemical basis of Mer2 association with Hop1, a HORMA domain-containing chromosomal axis factor. Finally, we identify a conserved region within Mer2 crucial for DSB activity, and show that this region of Mer2 interacts with the DSB factor Mre11. In combination with previous work, we establish Mer2 as a keystone of the DSB machinery by bridging key protein complexes involved in the initiation of meiotic recombination.

Organisms are said to be diploid when they carry two copies of each chromosome in their cells, one from each of their biological parents. But in order for each parent to only pass on one copy of their own chromosomes, they need to make haploid cells, which only carry one copy of each chromosome. These cells form by a special kind of cell division called meiosis, in which the two chromosomes from each pair in the parent cells are first linked, and then pulled apart into the daughter cells. Accurate meiosis requires a type of DNA damage called double-stranded DNA breaks. These breaks cut through the chromosomes and can be dangerous to the cell if they are not repaired correctly. During meiosis, a set of proteins gather around the chromosomes to ensure the cuts happen in the right place and to repair the damage. One of these proteins is called Mer2. Previous studies suggest that this protein plays a role in placing the DNA breaks and controlling when they happen. To find out more, Rousova et al. examined Mer2 and the proteins that interact with it in budding yeast cells. This involved taking the proteins out of the cell to get a closer look. The experiments showed that Mer2 sticks directly to the chromosomes and acts as a tether for other proteins. It collaborates with two partners, called Hop1 and Mre11, to make sure that DNA breaks happen safely. These proteins detect the state of the chromosome and repair the damage. Stopping Mer2 from interacting with Mre11 prevented DNA breaks from forming in budding yeast cells. Although Rousova et al. used budding yeast to study the proteins involved in meiosis, similar proteins exist in plant and animal cells too. Understanding how they work could open new avenues of research into cell division. For example, studies on plant proteins could provide tools for creating new crop strains. Studies on human proteins could also provide insights into fertility problems and cancer.

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

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.