Phosphorylation of ORC2 protein dissociates origin recognition complex from chromatin and replication origins

During the late M to the G(1) phase of the cell cycle, the origin recognition complex (ORC) binds to the replication origin, leading to the assembly of the prereplicative complex for subsequent initiation of eukaryotic chromosome replication. We found that the cell cycle-dependent phosphorylation of human ORC2, one of the six subunits of ORC, dissociates ORC2, -3, -4, and -5 (ORC2-5) subunits from chromatin and replication origins. Phosphorylation at Thr-116 and Thr-226 of ORC2 occurs by cyclin-dependent kinase during the S phase and is maintained until the M phase. Phosphorylation of ORC2 at Thr-116 and Thr-226 dissociated the ORC2-5 from chromatin. Consistent with this, the phosphomimetic ORC2 protein exhibited defective binding to replication origins as well as to chromatin, whereas the phosphodefective protein persisted in binding throughout the cell cycle. These results suggest that the phosphorylation of ORC2 dissociates ORC from chromatin and replication origins and inhibits binding of ORC to newly replicated DNA.     

Site-Specific DNA Binding of the Schizosaccharomyces pombe Origin Recognition Complex Is Determined by the Orc4 Subunit

The mechanism by which origin recognition complexes (ORCs) identify replication origins was investigated using purified Orc proteins from Schizosaccharomyces pombe. Orc4p alone bound tightly and specifically to several sites within S. pombe replication origins that are genetically required for origin activity. These sites consisted of clusters of A or T residues on one strand but were devoid of either alternating A and T residues or GC-rich sequences. Addition of a complex consisting of Orc1, -2, -3, -5, and -6 proteins (ORC-5) altered neither Orc4p binding to origin DNA nor Orc4p protection of specific sequences. ORC-5 alone bound weakly and nonspecifically to DNA; strong binding required the presence of Orc4p. Under these conditions, all six subunits remained bound to chromatin isolated from each phase of the cell division cycle. These results reveal that the S. pombe ORC binds to multiple, specific sites within replication origins and that site selection, at least in vitro, is determined solely by the Orc4p subunit.     

In vitro protein-DNA interactions at the human lamin B2 replication origin

The complexity of mammalian origins of DNA replication has prevented, so far, the in vitro studies of the modalities of initiator protein binding and origin selection. We approached this problem by utilizing the human lamin B2 origin, wherein the precise start sites of replication initiation have been identified and known to be bound in vivo by the origin recognition complex (ORC). In order to analyze the in vitro interactions occurring at this origin, we have compared the DNA binding requirements and patterns of the human recombinant Orc4 with those of preparations of HeLa nuclear proteins containing the ORC complex. Here we show that both HsOrc4 alone and HeLa nuclear proteins recognize multiple sites within a 241-bp DNA sequence encompassing the lamin B2 origin. The DNA binding activity of HeLa cells requires the presence of ORC and can be reproduced in the absence of all the other proteins known to be recruited to origins by ORC. Both HsOrc4 alone and HeLa nuclear proteins exhibit cooperative and ATP-independent binding. This binding covers nucleotides 3853-3953 and then spreads outward. Because this region contains the start sites of DNA synthesis as well as the area protected in vivo and preserves protein binding capacity in vitro after removal of a fraction of the protected region, we suggest that it could contain the primary binding site. Thus the in vitro approach points to the sequence requirements for ORC binding as a key element for origin recognition.     

Purification and characterization of the Schizosaccharomyces pombe origin recognition complex: interaction with origin DNA and Cdc18 protein

The origin recognition complex (ORC) plays a central role in the initiation of DNA replication in eukaryotic cells. It interacts with origins of DNA replication in chromosomal DNA and recruits additional replication proteins to form functional initiation complexes. These processes have not been well characterized at the biochemical level except in the case of Saccharomyces cerevisiae ORC. We report here the expression, purification, and initial characterization of Schizosaccharomyces pombe ORC (SpORC) containing six recombinant subunits. Purified SpORC binds efficiently to the ars1 origin of DNA replication via the essential Nterminal domain of the SpOrc4 subunit which contains nine AT-hook motifs. Competition binding experiments demonstrated that SpORC binds preferentially to DNA molecules rich in AT-tracts, but does not otherwise exhibit a high degree of sequence specificity. The complex is capable of binding to multiple sites within the ars1 origin of DNA replication with similar affinities, indicating that the sequence requirements for origin recognition in S. pombe are significantly less stringent than in S. cerevisiae. We have also demonstrated that SpORC interacts directly with Cdc18p, an essential fission yeast initiation protein, and recruits it to the ars1 origin in vitro. Recruitment of Cdc18p to chromosomal origins is a likely early step in the initiation of DNA replication in vivo. These data indicate that the purified recombinant SpORC retains at least two of its primary biological functions and that it will be useful for the eventual reconstitution of the initiation reaction with purified proteins.     

Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila melanogaster

The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of the Orc6 protein which does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.     

Nucleosomes positioned by ORC facilitate the initiation of DNA replication

The packaging of eukaryotic DNA into nucleosomes is a critical regulator of nuclear events. To address the interplay between chromatin and replication initiation, we have assessed the determinants and function of the nucleosomal configuration of S. cerevisiae replication origins. Using in vitro and in vivo assays, we demonstrate that the yeast initiator, the origin recognition complex (ORC), is required to maintain the nucleosomal configuration adjacent to origins. Disruption of the ORC-directed nucleosomal arrangement at an origin interferes with initiation of replication, but does not alter the association of ORC with the origin. Instead, the nucleosomes positioned by ORC are important for prereplicative complex formation. These findings suggest that origin-proximal nucleosomes facilitate replication initiation, and that local chromatin structure affects origin function.     

Human Origin Recognition Complex Binds Preferentially to G-quadruplex-preferable RNA and Single-stranded DNA

Origin recognition complex (ORC), consisting of six subunits ORC1–6, is known to bind to replication origins and function in the initiation of DNA replication in eukaryotic cells. In contrast to the fact that Saccharomyces cerevisiae ORC recognizes the replication origin in a sequence-specific manner, metazoan ORC has not exhibited strict sequence-specificity for DNA binding. Here we report that human ORC binds preferentially to G-quadruplex (G4)-preferable G-rich RNA or single-stranded DNA (ssDNA). We mapped the G-rich RNA-binding domain in the ORC1 subunit, in a region adjacent to its ATPase domain. This domain itself has an ability to preferentially recognize G4-preferable sequences of ssDNA. Furthermore, we found, by structure modeling, that the G-rich RNA-binding domain is similar to the N-terminal portion of AdoMet_MTase domain of mammalian DNA methyltransferase 1. Therefore, in contrast with the binding to double-stranded DNA, human ORC has an apparent sequence preference with respect to its RNA/ssDNA binding. Interestingly, this specificity coincides with the common signature present in most of the human replication origins. We expect that our findings provide new insights into the regulations of function and chromatin binding of metazoan ORCs.     

ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA

Binding of Cdc6 to the origin recognition complex (ORC) is a key step in the assembly of a pre-replication complex (pre-RC) at origins of DNA replication. ORC recognizes specific origin DNA sequences in an ATP-dependent manner. Here we demonstrate cooperative binding of Saccharomyces cerevisiae Cdc6 to ORC on DNA in an ATP-dependent manner, which induces a change in the pattern of origin binding that requires the Orc1 ATPase. The reaction is blocked by specific origin mutations that do not interfere with the interaction between ORC and DNA. Single-particle reconstruction of electron microscopic images shows that the ORC-Cdc6 complex forms a ring-shaped structure with dimensions similar to those of the ring-shaped MCM helicase. The ORC-Cdc6 structure is predicted to contain six AAA+ subunits, analogous to other ATP-dependent protein machines. We suggest that Cdc6 and origin DNA activate a molecular switch in ORC that contributes to pre-RC assembly.     

DNA sequence templates adjacent nucleosome and ORC sites at gene amplification origins in Drosophila

Eukaryotic origins of DNA replication are bound by the origin recognition complex (ORC), which scaffolds assembly of a pre-replicative complex (pre-RC) that is then activated to initiate replication. Both pre-RC assembly and activation are strongly influenced by developmental changes to the epigenome, but molecular mechanisms remain incompletely defined. We have been examining the activation of origins responsible for developmental gene amplification in Drosophila. At a specific time in oogenesis, somatic follicle cells transition from genomic replication to a locus-specific replication from six amplicon origins. Previous evidence indicated that these amplicon origins are activated by nucleosome acetylation, but how this affects origin chromatin is unknown. Here, we examine nucleosome position in follicle cells using micrococcal nuclease digestion with Ilumina sequencing. The results indicate that ORC binding sites and other essential origin sequences are nucleosome-depleted regions (NDRs). Nucleosome position at the amplicons was highly similar among developmental stages during which ORC is or is not bound, indicating that being an NDR is not sufficient to specify ORC binding. Importantly, the data suggest that nucleosomes and ORC have opposite preferences for DNA sequence and structure. We propose that nucleosome hyperacetylation promotes pre-RC assembly onto adjacent DNA sequences that are disfavored by nucleosomes but favored by ORC.     

Eukaryotic origins of DNA replication: could you please be more specific?

Initiation of eukaryotic DNA replication commences when the origin recognition complex (ORC) binds to DNA, recruiting helicases, polymerases, and necessary cofactors. While the biochemical mechanism and factors involved in replication initiation appear to be highly conserved, the DNA sequences at which these events take place in different organisms are not. Thus, while ORC appears to bind to specific DNA sequences in budding yeast, there is increasing new evidence that metazoan ORC complexes do not rely on sequence to be directed to origins of replication. Here, we review examples of specific and non-specific initiation, and we consider what, if not DNA sequence, accounts for DNA binding of ORC to defined regions in eukaryotic genomes.     

Control of ATP-Dependent Binding of Saccharomyces cerevisiae Origin Recognition Complex to Autonomously Replicating DNA Sequences

Eukaryotic origin recognition complexes (ORCs) play pivotal roles in the initiation of chromosomal DNA replication. ORC from the yeast, Saccharomyces cerevisiae, recognizes and binds replication origins in the late G1 phase and the binding has profound implications in the progression of the cell cycle to the S-phase. Therefore, we have quantitatively analyzed the mechanism of recognition and interaction of the yeast ORC with various elements of a yeast origin of DNA replication, the autonomously replicating sequence 1 (ARS1). ORC bound all four individual A and B elements of ARS1 with reasonably high affinities. However, the highest affinity binding was observed with a DNA sequence containing both the A and B1 elements. In addition, ATP and ADP significantly modulated the binding of ORC to the combined elements as well as modulating the binding of ORC to the element A alone or in combination with the B1 element. However, binding of ORC to individual B1, B2, and B3 elements was not responsive to nucleotides. Thus, the consensus ARS sequence in element A appeared to play a pivotal role in the ATP-dependent binding of ORC to ARS1 and likely in other ARSs or origins of DNA replication.     

The spatial arrangement of ORC binding modules determines the functionality of replication origins in budding yeast

In the quest to define autonomously replicating sequences (ARSs) in eukaryotic cells, an ARS consensus sequence (ACS) has emerged for budding yeast. This ACS is recognized by the replication initiator, the origin recognition complex (ORC). However, not every match to the ACS constitutes a replication origin. Here, we investigated the requirements for ORC binding to origins that carry multiple, redundant ACSs, such as ARS603. Previous studies raised the possibility that these ACSs function as individual ORC binding sites. Detailed mutational analysis of the two ACSs in ARS603 revealed that they function in concert and give rise to an initiation pattern compatible with a single bipartite ORC binding site. Consistent with this notion, deletion of one base pair between the ACS matches abolished ORC binding at ARS603. Importantly, loss of ORC binding in vitro correlated with the loss of ARS activity in vivo. Our results argue that replication origins in yeast are in general comprised of bipartite ORC binding sites that cannot function in random alignment but must conform to a configuration that permits ORC binding. These requirements help to explain why only a limited number of ACS matches in the yeast genome qualify as ORC binding sites.     

Identification of ORC1/CDC6-interacting factors in Trypanosoma brucei reveals critical features of origin recognition complex architecture

DNA replication initiates by formation of a pre-replication complex on sequences termed origins. In eukaryotes, the pre-replication complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1-6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing.     

The dynamics of replication licensing in live Caenorhabditis elegans embryos

Accurate DNA replication requires proper regulation of replication licensing, which entails loading MCM-2-7 onto replication origins. In this paper, we provide the first comprehensive view of replication licensing in vivo, using video microscopy of Caenorhabditis elegans embryos. As expected, MCM-2-7 loading in late M phase depended on the prereplicative complex (pre-RC) proteins: origin recognition complex (ORC), CDC-6, and CDT-1. However, many features we observed have not been described before: GFP-ORC-1 bound chromatin independently of ORC-2-5, and CDC-6 bound chromatin independently of ORC, whereas CDT-1 and MCM-2-7 DNA binding was interdependent. MCM-3 chromatin loading was irreversible, but CDC-6 and ORC turned over rapidly, consistent with ORC/CDC-6 loading multiple MCM-2-7 complexes. MCM-2-7 chromatin loading further reduced ORC and CDC-6 DNA binding. This dynamic behavior creates a feedback loop allowing ORC/CDC-6 to repeatedly load MCM-2-7 and distribute licensed origins along chromosomal DNA. During S phase, ORC and CDC-6 were excluded from nuclei, and DNA was overreplicated in export-defective cells. Thus, nucleocytoplasmic compartmentalization of licensing factors ensures that DNA replication occurs only once.     

RNA-dependent recruitment of the origin recognition complex

The origin recognition complex (ORC) has an important function in determining the initiation sites of DNA replication. In higher eukaryotes, ORC lacks sequence-specific DNA binding, and the mechanisms of ORC recruitment and origin determination are poorly understood. ORC is recruited with high efficiency to the Epstein-Barr virus origin of plasmid replication (OriP) through a complex mechanism involving interactions with the virus-encoded EBNA1 protein. We present evidence that ORC recruitment to OriP and DNA replication function depends on RGG-like motifs, referred to as LR1 and LR2, in the EBNA1 amino-terminal domain. Moreover, we show that LR1 and LR2 recruitment of ORC is RNA dependent. HMGA1a, which can functionally substitute for LR1 and LR2 domain, can also recruit ORC in an RNA-dependent manner. EBNA1 and HMGA1a RGG motifs bound to structured G-rich RNA, as did ORC1 peptides, which interact with EBNA1. RNase A treatment of cellular chromatin released a fraction of the total ORC, suggesting that ORC association with chromatin, and possibly cellular origins, is stabilized by RNA. We propose that structural RNA molecules mediate ORC recruitment at some cellular and viral origins, similar to OriP.     

Cdc6 ATPase activity regulates ORC x Cdc6 stability and the selection of specific DNA sequences as origins of DNA replication

DNA replication, as with all macromolecular synthesis steps, is controlled in part at the level of initiation. Although the origin recognition complex (ORC) binds to origins of DNA replication, it does not solely determine their location. To initiate DNA replication ORC requires Cdc6 to target initiation to specific DNA sequences in chromosomes and with Cdt1 loads the ring-shaped mini-chromosome maintenance (MCM) 2-7 DNA helicase component onto DNA. ORC and Cdc6 combine to form a ring-shaped complex that contains six AAA+ subunits. ORC and Cdc6 ATPase mutants are defective in MCM loading, and ORC ATPase mutants have reduced activity in ORC x Cdc6 x DNA complex formation. Here we analyzed the role of the Cdc6 ATPase on ORC x Cdc6 complex stability in the presence or absence of specific DNA sequences. Cdc6 ATPase is activated by ORC, regulates ORC x Cdc6 complex stability, and is suppressed by origin DNA. Mutations in the conserved origin A element, and to a lesser extent mutations in the B1 and B2 elements, induce Cdc6 ATPase activity and prevent stable ORC x Cdc6 formation. By analyzing ORC x Cdc6 complex stability on various DNAs, we demonstrated that specific DNA sequences control the rate of Cdc6 ATPase, which in turn controls the rate of Cdc6 dissociation from the ORC x Cdc6 x DNA complex. We propose a mechanism explaining how Cdc6 ATPase activity promotes origin DNA sequence specificity; on DNA that lacks origin activity, Cdc6 ATPase promotes dissociation of Cdc6, whereas origin DNA down-regulates Cdc6 ATPase resulting in a stable ORC x Cdc6 x DNA complex, which can then promote MCM loading. This model has relevance for origin specificity in higher eukaryotes.     

Mapping subunit location on the Saccharomyces cerevisiae origin recognition complex free and bound to DNA using a novel nanoscale biopointer

The Saccharomyces cerevisiae origin recognition complex (ORC) is composed of six subunits and is an essential component in the assembly of the replication apparatus. To probe the organization of this multiprotein complex by electron microscopy, each subunit was tagged on either its C or N terminus with biotin and assembled into a complex with the five other unmodified subunits. A nanoscale biopointer consisting of a short DNA duplex with streptavidin at one end was used to map the location of the N and C termini of each subunit. These observations were made using ORC free in solution and bound to the ARS1 origin of replication. This mapping confirms and extends previous studies mapping the sites of subunit interaction with origin DNA. In particular, we provide new information concerning the stoichiometry of the ORC-ARS1 complex and the changes in conformation that are associated with DNA binding by ORC. This versatile, new approach to mapping protein structure has potential for many applications.     

A human cancer cell line initiates DNA replication normally in the absence of ORC5 and ORC2 proteins

The origin recognition complex (ORC), composed of six subunits, ORC1–6, binds to origins of replication as a ring-shaped heterohexameric ATPase that is believed to be essential to recruit and load MCM2–7, the minichromosome maintenance protein complex, around DNA and initiate DNA replication. We previously reported the creation of viable cancer cell lines that lacked detectable ORC1 or ORC2 protein without a reduction in the number of origins firing. Here, using CRISPR-Cas9–mediated mutations, we report that human HCT116 colon cancer cells also survive when ORC5 protein expression is abolished via a mutation in the initiator ATG of the ORC5 gene. Even if an internal methionine is used to produce an undetectable, N terminally deleted ORC5, the protein would lack 80% of the AAA+ ATPase domain, including the Walker A motif. The ORC5-depleted cells show normal chromatin binding of MCM2–7 and initiate replication from a similar number of origins as WT cells. In addition, we introduced a second mutation in ORC2 in the ORC5 mutant cells, rendering both ORC5 and ORC2 proteins undetectable in the same cells and destabilizing the ORC1, ORC3, and ORC4 proteins. Yet the double mutant cells grow, recruit MCM2–7 normally to chromatin, and initiate DNA replication with normal number of origins. Thus, in these selected cancer cells, either a crippled ORC lacking ORC2 and ORC5 and present at minimal levels on the chromatin can recruit and load enough MCM2–7 to initiate DNA replication, or human cell lines can sometimes recruit MCM2–7 to origins independent of ORC.     

Diverged composition and regulation of the Trypanosoma brucei origin recognition complex that mediates DNA replication initiation

Initiation of DNA replication depends upon recognition of genomic sites, termed origins, by AAA+ ATPases. In prokaryotes a single factor binds each origin, whereas in eukaryotes this role is played by a six-protein origin recognition complex (ORC). Why eukaryotes evolved a multisubunit initiator, and the roles of each component, remains unclear. In Trypanosoma brucei, an ancient unicellular eukaryote, only one ORC-related initiator, TbORC1/CDC6, has been identified by sequence homology. Here we show that three TbORC1/CDC6-interacting factors also act in T. brucei nuclear DNA replication and demonstrate that TbORC1/CDC6 interacts in a high molecular complex in which a diverged Orc4 homologue and one replicative helicase subunit can also be found. Analysing the subcellular localization of four TbORC1/CDC6-interacting factors during the cell cycle reveals that one factor, TbORC1B, is not a static constituent of ORC but displays S-phase restricted nuclear localization and expression, suggesting it positively regulates replication. This work shows that ORC architecture and regulation are diverged features of DNA replication initiation in T. brucei, providing new insight into this key stage of eukaryotic genome copying.