Michael R. Botchan

Summary: The Origin Recognition Complex (ORC) is known for initiating DNA replication in eukaryotic cells, but increasing evidence suggests that ORC has multiple functions. Here we show that the organization and function of two major nuclear components, heterochromatin and the nucleolus, depend on multivalent interactions between Orc1 and Heterochromatin Protein 1a (HP1a) in Drosophila melanogaster . Specifically, binding requires two short motifs (R1 and R2) in the intrinsically disordered region (IDR) of Orc1, and two motifs (HGM and CTE) located in the N- and C-terminal regions of HP1a. Pairing of these four motifs promotes ORC–HP1 interactions, which also requires HP1 dimerization. Disrupting ORC–HP1a interactions by mutating the R1/R2 motifs causes defects in heterochromatin functions, specifically suppression of Position-Effect Variegation (PEV), ribosomal DNA (rDNA) decondensation, increased rRNA transcription, and nucleolar expansion, without global loss of H3K9me2 epigenetic modifications. Our findings indicate that ORC acts as a structural regulator that organizes heterochromatic regions and promotes proper genome regulation through multivalent interactions with HP1 and chromatin.

Liquid–liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. Previously, we showed that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo, and that initiator condensates selectively recruit replication-specific partner proteins (Parker et al., 2019). How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. Here, using Drosophila melanogaster (Dm) Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6-hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive condensate formation and specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and partner selection a priori.

Liquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. We have shown that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo , and that initiator condensates selectively recruit specific partner proteins. How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. Using D. melanogaster (Dm) Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6- hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive self-assembly and condensate specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and specificity a priori .

The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the Mcm2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and Mcm2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.

SUMMARY: The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the Mcm2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro . We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and Mcm2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.

Significance: The onset of chromosomal DNA replication relies on dedicated initiator proteins to chaperone ring-shaped helicases onto DNA. In most eukaryotes, initiators are multisubunit protein complexes that require ATP to bind DNA and to aid helicase recruitment and loading. Although structural studies have recently elucidated high-resolution views of the initiator in isolation or in helicase-containing loading intermediates, how the eukaryotic initiator itself associates with DNA and how these interactions are regulated by conformational changes are not well understood. We use a combination of biochemical and structural studies of the Drosophila initiator origin recognition complex (ORC) to show that conformational alterations in metazoan ORC help regulate its DNA-binding activity, and that ORC, together with its cofactor Cdc6, bends substrate DNA prior to helicase loading.

Diverse molecular choreography of replication: Accurate duplication and transmission of genetic information to the next generation requires complex molecular assemblies. Bleichert et al. review replication initiation across the three domains of life, with a focus on origin selection and helicase loading. These processes identify potential origins of replication and prepare them for subsequent bidirectional replication initiation. There are key similarities and multiple differences in replication mechanisms between eukaryotes, prokaryotes, and archaea and many outstanding questions to be answered. Science , this issue p. eaah6317

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation – from selecting replication start sites to replicative helicase loading and activation – and describe how these events are often distinctly regulated across different eukaryotic model organisms.

Initiation of cellular DNA replication is tightly controlled to sustain genomic integrity. In eukaryotes, the heterohexameric origin recognition complex (ORC) is essential for coordinating replication onset. Here we describe the crystal structure of Drosophila ORC at 3.5 Å resolution, showing that the 270 kilodalton initiator core complex comprises a two-layered notched ring in which a collar of winged-helix domains from the Orc1–5 subunits sits atop a layer of AAA+ (ATPases associated with a variety of cellular activities) folds. Although canonical inter-AAA+ domain interactions exist between four of the six ORC subunits, unanticipated features are also evident. These include highly interdigitated domain-swapping interactions between the winged-helix folds and AAA+ modules of neighbouring protomers, and a quasi-spiral arrangement of DNA binding elements that circumnavigate an approximately 20 Å wide channel in the centre of the complex. Comparative analyses indicate that ORC encircles DNA, using its winged-helix domain face to engage the mini-chromosome maintenance 2–7 (MCM2–7) complex during replicative helicase loading; however, an observed out-of-plane rotation of more than 90° for the Orc1 AAA+ domain disrupts interactions with catalytic amino acids in Orc4, narrowing and sealing off entry into the central channel. Prima facie, our data indicate that Drosophila ORC can switch between active and autoinhibited conformations, suggesting a novel means for cell cycle and/or developmental control of ORC functions.

Significance: Cell division control protein 45 (Cdc45), a RecJ homologue, is essential in all eukaryotes. Cdc45 functions with the replisome CMG helicase where minichromosome maintenance (Mcm2–7) proteins provide motor activity for unwinding duplex during replication. We report that the dynamic gate between Mcm subunits 2 and 5, which is essential for the initial loading of the motor, may be an Achilles heel because the leading strand may slip from its central channel in an open gate state. Studies show that the side channel formed by the Cdc45 and GINS works as a trap and guards this gate; the Recombination protein J fold is key for this activity. We propose that this new function for Cdc45 will be important for fork integrity during the S-phase in response to double-strand breaks or replication stress.