Six-subunit Origin Recognition Complex Research Articles

Structural basis of DNA replication origin recognition by human Orc6 protein binding with DNA.

The six-subunit origin recognition complex (ORC), a DNA replication initiator, defines the localization of the origins of replication in eukaryotes. The Orc6 subunit is the smallest and the least conserved among ORC subunits. It is required for DNA replication and essential for viability in all species. Orc6 in metazoans carries a structural homology with transcription factor TFIIB and can bind DNA on its own. Here, we report a solution structure of the full-length human Orc6 (HsOrc6) alone and in a complex with DNA. We further showed that human Orc6 is composed of three independent domains: N-terminal, middle and C-terminal (HsOrc6-N, HsOrc6-M and HsOrc6-C). We also identified a distinct DNA-binding domain of human Orc6, named as HsOrc6-DBD. The detailed analysis of the structure revealed novel amino acid clusters important for the interaction with DNA. Alterations of these amino acids abolish DNA-binding ability of Orc6 and result in reduced levels of DNA replication. We propose that Orc6 is a DNA-binding subunit of human/metazoan ORC and may play roles in targeting, positioning and assembling the functional ORC at the origins.

Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6

DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M-phase of the cell-cycle, Cdc6 binds to ORC and the ORC-Cdc6 complex loads in a multistep reaction and, with the help of Cdt1, the core Mcm2-7 helicase onto DNA. A key intermediate is the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) complex in which DNA has been already inserted into the central channel of Mcm2-7. Until now, it has been unclear how the origin DNA is guided by ORC-Cdc6 and inserted into the Mcm2-7 hexamer. Here, we truncated the C-terminal winged-helix-domain (WHD) of Mcm6 to slow down the loading reaction, thereby capturing two loading intermediates prior to DNA insertion in budding yeast. In "semi-attached OCCM," the Mcm3 and Mcm7 WHDs latch onto ORC-Cdc6 while the main body of the Mcm2-7 hexamer is not connected. In "pre-insertion OCCM," the main body of Mcm2-7 docks onto ORC-Cdc6, and the origin DNA is bent and positioned adjacent to the open DNA entry gate, poised for insertion, at the Mcm2-Mcm5 interface. We used molecular simulations to reveal the dynamic transition from preloading conformers to the loaded conformers in which the loading of Mcm2-7 on DNA is complete and the DNA entry gate is fully closed. Our work provides multiple molecular insights into a key event of eukaryotic DNA replication.

Structure of the origin recognition complex bound to DNA replication origin.

The six-subunit origin recognition complex (ORC) binds to DNA to mark the site for the initiation of replication in eukaryotes. Here we report a 3Å cryo-electron microscopy structure of the Saccharomyces cerevisiae ORC bound to a 72-base-pair origin DNA sequence that contains the ARS consensus sequence (ACS) and the B1 element. The ORC encircles DNA through extensive interactions with both phosphate backbone and bases, and bends DNA at the ACS and B1 sites. Specific recognition of thymine residues in the ACS is carried out by a conserved basic amino acid motif of Orc1 in the minor groove, and by a species-specific helical insertion motif of Orc4 in the major groove. Moreover, similar insertions into major and minor grooves are also embedded in the B1 site by basic patch motifs from Orc2 and Orc5, respectively, to contact bases and to bend DNA. This work pinpoints a conserved role of ORC in modulating DNA structure to facilitate origin selection and helicase loading in eukaryotes.

Two subunits of human ORC are dispensable for DNA replication and proliferation.

The six-subunit Origin Recognition Complex (ORC) is believed to be an essential eukaryotic ATPase that binds to origins of replication as a ring-shaped heterohexamer to load MCM2-7 and initiate DNA replication. We have discovered that human cell lines in culture proliferate with intact chromosomal origins of replication after disruption of both alleles of ORC2 or of the ATPase subunit, ORC1. The ORC1 or ORC2-depleted cells replicate with decreased chromatin loading of MCM2-7 and become critically dependent on another ATPase, CDC6, for survival and DNA replication. Thus, either the ORC ring lacking a subunit, even its ATPase subunit, can load enough MCM2-7 in partnership with CDC6 to initiate DNA replication, or cells have an ORC-independent, CDC6-dependent mechanism to load MCM2-7 on origins of replication.

Concerted interaction between origin recognition complex (ORC), nucleosomes and replication originDNAensures stableORC–origin binding

Chromosomal replication origins, where DNA replication is initiated, are determined in eukaryotic cells by specific binding of a six-subunit origin recognition complex (ORC). Many biochemical analyses have showed the detailed properties of the ORC-DNA interaction. However, because of the lack of in vitro analysis, the molecular architecture of the ORC-chromatin interaction is unclear. Recently, mainly from in vivo analyses, a role of chromatin in the ORC-origin interaction has been reported, including the existence of a specific pattern of nucleosome positioning around origins and of a specific interaction between chromatin-or core histones-and Orc1, a subunit of ORC. Therefore, to understand how ORC establishes its interaction with origin in vivo, it is essential to know the molecular mechanisms of the ORC-chromatin interaction. Here, we show that ORC purified from yeast binds more stably to origin-containing reconstituted chromatin than to naked DNA and forms a nucleosome-free region at origins. Molecular imaging using atomic force microscopy (AFM) shows that ORC associates with the adjacent nucleosomes and forms a larger complex. Moreover, stable binding of ORC to chromatin requires linker DNA. Thus, ORC establishes its interaction with origin by binding to both nucleosome-free origin DNA and neighboring nucleosomes.

Assembly of the Human Origin Recognition Complex Occurs through Independent Nuclear Localization of Its Components

Initiation of eukaryotic genome duplication begins when a six-subunit origin recognition complex (ORC) binds to DNA. However, the mechanism by which this occurs in vivo and the roles played by individual subunits appear to differ significantly among organisms. Previous studies identified a soluble human ORC(2-5) complex in the nucleus, an ORC(1-5) complex bound to chromatin, and an Orc6 protein that binds weakly, if at all, to other ORC subunits. Here we show that stable ORC(1-6) complexes also can be purified from human cell extracts and that Orc6 and Orc1 each contain a single nuclear localization signal that is essential for nuclear localization but not for ORC assembly. The Orc6 nuclear localization signal, which is essential for Orc6 function, is facilitated by phosphorylation at its cyclin-dependent kinase consensus site and by association with Kpna6/1, nuclear transport proteins that did not co-purify with other ORC subunits. These and other results support a model in which Orc6, Orc1, and ORC(2-5) are transported independently to the nucleus where they can either assemble into ORC(1-6) or function individually.

Different roles of the human Orc6 protein in the replication initiation process.

In eukaryotes, binding of the six-subunit origin recognition complex (ORC) to DNA provides an interactive platform for the sequential assembly of pre-replicative complexes. This process licenses replication origins competent for the subsequent initiation step. Here, we analyze the contribution of human Orc6, the smallest subunit of ORC, to DNA binding and pre-replicative complex formation. We show that Orc6 not only interacts with Orc1-Orc5 but also with the initiation factor Cdc6. Biochemical and imaging experiments reveal that this interaction is required for licensing DNA replication competent. Furthermore, we demonstrate that Orc6 contributes to the interaction of ORC with the chaperone protein HMGA1a (high mobility group protein A1a). Binding of human ORC to replication origins is not specified at the level of DNA sequence and the functional organization of origins is poorly understood. We have identified HMGA1a as one factor that might direct ORC to AT-rich heterochromatic regions. The systematic analysis of the interaction between ORC and HMGA1a revealed that Orc6 interacts with the acidic C-terminus of HMGA1a and also with its AT-hooks. Both domains support autonomous replication if targeted to DNA templates. As such, Orc6 functions at different stages of the replication initiation process. Orc6 can interact with ORC chaperone proteins such as HMGA1a to facilitate chromatin binding of ORC and is also an essential factor for pre-RC formation.

Positive roles of SAS2 in DNA replication and transcriptional silencing in yeast

Sas2p is a histone acetyltransferase implicated in the regulation of transcriptional silencing, and ORC is the six-subunit origin recognition complex involved in the initiation of DNA replication and the establishment of transcriptionally silent chromatin by silencers in yeast. We show here that SAS2 deletion (sas2Δ) exacerbates the temperature sensitivity of the ORC mutants orc2-1 and orc5-1. Moreover, sas2Δ and orc2-1 have a synthetic effect on cell cycle progression through S phase and initiation of DNA replication. These results suggest that SAS2 plays a positive role in DNA replication and cell cycle progression. We also show that sas2Δ and orc5-1 have a synthetic effect on transcriptional silencing at the HMR locus. Moreover, we demonstrate that sas2Δ reduces the silencing activities of silencers regardless of their locations and contexts, indicating that SAS2 plays a positive role in silencer function. In addition, we show that SAS2 is required for maintaining the structure of transcriptionally silent chromatin.

Schizosaccharomyces pombe Orc5 plays multiple roles in the maintenance of genome stability throughout the cell cycle

The six-subunit origin recognition complex (ORC) acts as a landing pad for factors that initiate DNA replication by binding to replication origins. In addition, ORC is involved in other processes such as transcriptional gene silencing and sister chromatid cohesion in Saccharomyces cerevisiae. However, whether these functions of ORC are specific to Saccharomyces cerevisiae or are shared by the ORC of other organisms is currently unclear. Analysis of two temperature-sensitive alleles of the fifth ORC subunit of Saccharomyces pombe, orc5-H19 and orc5-H37, indicates that Orc5 of Saccharomyces pombe has similar multiple functions to those of Orc5 of Saccharomyces cerevisiae. The orc5-H19 cells were defective in DNA replication initiation, and execution point analysis of this mutant revealed that ORC functions before metaphase to prepare for the initiation of replication in the next cell cycle. The orc5-H37 cells seemed to complete DNA synthesis but were arrested before entering M phase. In both mutants, the rads-chk1 checkpoint was activated to prevent mitosis, suggesting that this checkpoint pathway monitors the functional integrity of ORC. In addition, orc5-H37 cells showed premature separation of sister chromatids, which resulted in cell growth being dependent on the mad2-dependent spindle checkpoint. Consistently, this mutant showed a defect in the loading of Rad21, a cohesin component. Based on these observations, we propose that Orc5 has at least two distinct functions that can be separated genetically. Taken together, our results provide further support for the idea that ORC plays multiple functions during the cell cycle.

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.

Degradation of Cdt1 during S Phase Is Skp2-independent and Is Required for Efficient Progression of Mammalian Cells through S Phase

Previous reports have shown that the N terminus of Cdt1 is required for its degradation during S phase (Li, X., Zhao, Q., Liao, R., Sun, P., and Wu, X. (2003) J. Biol. Chem. 278, 30854-30858; Nishitani, H., Lygerou, Z., and Nishimoto, T. (2004) J. Biol. Chem. 279, 30807-30816). The stabilization was attributed to deletion of the cyclin binding motif (Cy motif), which is required for its phosphorylation by cyclin-dependent kinases. Phosphorylated Cdt1 is subsequently recognized by the F-box protein Skp2 and targeted for proteasomal mediated degradation. Using phosphopeptide mapping and mutagenesis studies, we found that threonine 29 within the N terminus of Cdt1 is phosphorylated by Cdk2 and required for interaction with Skp2. However, threonine 29 and the Cy motif are not necessary for proteolysis of Cdt1 during S phase. Mutants of Cdt1 that do not stably associate with Skp2 or cyclins are still degraded in S phase to the same extent as wild type Cdt1, indicating that other determinants within the N terminus of Cdt1 are required for degrading Cdt1. We localized the region necessary for Cdt1 degradation to the first 32 residues. Overexpression of stable forms of Cdt1 significantly delayed entry into and completion of S phase, suggesting that failure to degrade Cdt1 prevents normal progression through S phase. In contrast, Cdt1 mutants that fail to interact with Skp2 and cyclins progress through S phase with similar kinetics as wild type Cdt1 but stimulate the re-replication caused by overexpressing Cdt1. Therefore, a Skp2-independent pathway that requires the N-terminal 32 residues of Cdt1 is critical for the degradation of Cdt1 in S phase, and this degradation is necessary for the optimum progression of cells through S phase.

Studies of the properties of human origin recognition complex and its Walker A motif mutants

The eukaryotic six-subunit origin recognition complex (ORC) governs the initiation site of DNA replication and formation of the prereplication complex. In this report we describe the isolation of the wild-type Homo sapiens (Hs)ORC and variants containing a Walker A motif mutation in the Orc1, Orc4, or Orc5 subunit using the baculovirus-expression system. Coexpression of all six HsORC subunits yielded a stable complex containing HsOrc subunits 1-5 (HsORC1-5) with virtually no Orc6 protein (Orc6p). We examined the ATPase, DNA-binding, and replication activities of these complexes. Similar to other eukaryotic ORCs, wild-type HsORC1-5 possesses ATPase activity that is stimulated only 2-fold by single-stranded DNA. HsORC1-5 with a mutated Walker A motif in Orc1p contains no ATPase activity, whereas a similar mutation of either the Orc4 or Orc5 subunit did not affect this activity. The DNA-binding activity of HsORC1-5, using lamin B2 DNA as substrate, is stimulated by ATP 3- to 5-fold. Mutations in the Walker A motif of Orc1p, Orc4p, or Orc5p reduced the binding efficiency of HsORC1-5 modestly (2- to 5-fold). Xenopus laevis ORC-depleted extracts supplemented with HsORC1-5 supported prereplication complex formation and X. laevis sperm DNA replication, whereas the complex with a mutation in the Walker A motif of the Orc1, Orc4, or Orc5 subunit did not. These studies indicate that the ATP-binding motifs of Orc1, Orc4, and Orc5 are all essential for the replication activity associated with HsORC.

The 3 Rs of Cdc6: Recruitment, Regulation, and Replication

What is it? The cell division cycle genes CDC6 and CDC18 encode budding and fission yeast orthologs identified in a series of elegant screens for regulators of cell cycle progression performed independently by Leland Hartwell and Paul Nurse in the 1970s. In the subsequent three decades, orthologs have been found throughout Eukaryota and have been revealed to play key roles in two fundamental aspects of replication, recruiting replication components to chromosomes, and helping safeguard against multiple rounds of DNA replication.How does it do both? Cdc6 is part of a macro-molecular machine that assembles on chromatin to license the DNA for a single round of replication. Cdc6 collaborates with the six-subunit origin recognition complex (ORC) and Cdt1 to recruit the minichromosome maintenance (MCM) family of proteins to DNA, thereby forming the prereplication complex (see Figure 1Figure 1). Once the DNA has been licensed, S phase promoting factor, which consists of various regulatory kinases including Cdc7 and cyclin-dependent kinases (CDKs), triggers the prereplication complexes to fire. In licensing the DNA, Cdc6 simultaneously lays the groundwork for ensuring each segment of the chromosomal DNA is replicated only once each cell cycle: concomitant with origin firing, Cdc6 disassembles from the prereplication complex, thereby blocking rereplication. Cdc6 is thus part of a unidirectional switch at replication origins that is triggered only once per S phase.Figure 1Initiation of eukaryotic DNA replication. During G1 phase, ORC (composed of six subunits), Cdc6 (which likely acts as a multimer), Cdt1, and MCMs (a ring-shaped clamp composed of six subunits) assemble on chromosomes to form the prereplication complex. During S phase, CDK phosphorylates Cdc6, which in turn is either degraded by SCF (yeast) or relocalized to the cytoplasm (metazoans).View Large Image | View Hi-Res Image | Download PowerPoint SlideIs Cdc6 a clamp loader? A clamp loader is a protein complex that uses ATP binding and hydrolysis as molecular switches to load a ring-shaped DNA processivity factor, the clamp, onto DNA. Biochemical analyses of eukaryotic Cdc6 suggest that this is just how Cdc6 works. That is, Cdc6 likely acts as a multimer in which ATP binding and hydrolysis induce conformational changes that result in the recruitment of MCM – a putative ring-shaped clamp – to DNA (see Figure 1Figure 1). Despite having limited sequence similarity, the three-dimensional structures of clamp loaders are remarkably similar among prokaryotic, archaeal and eukaryotic organisms, and indeed, an archaeal Cdc6 ortholog has recently been shown to contain clamp loader structural elements.How is Cdc6 regulated? In addition to changes in activity via interaction with ATP, Cdc6 is regulated by changes in its abundance and localization. Yeast and metazoans regulate Cdc6 through CDK-mediated phosphorylation during early S phase. In yeast, phosphorylated Cdc6 is subsequently degraded in a Skp1, Cdc53/Cullin, F box receptor (SCF)-dependent ubiquitin-mediated pathway. In contrast, phosphorylation of mammalian Cdc6 promotes its export from the nucleus into the cytoplasm where it is ubiquitinated by the anaphase-promoting complex (APC) and degraded during mitosis (see Figure 1Figure 1). In fission yeast, overexpression of Cdc18 causes endoreduplication without an intervening mitosis. However, similar mutations that override the degradation or localization controls of Cdc6 in either budding yeast or metazoans are not sufficient to overcome the block to rereplication. In fact, recent studies in budding yeast indicate that regulation of Cdc6 is one of several redundant mechanisms that prevent rereplication in eukaryotic cells.

Assembly of the Human Origin Recognition Complex

The six-subunit origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae. Yeast ORC binds specifically to origins of replication and serves as a platform for the assembly of additional initiation factors, such as Cdc6 and the Mcm proteins. Human homologues of all six ORC subunits have been identified by sequence similarity to their yeast counterparts, but little is known about the biochemical characteristics of human ORC (HsORC). We have extracted HsORC from HeLa cell chromatin and probed its subunit composition using specific antibodies. The endogenous HsORC, identified in these experiments, contained homologues of Orc1-Orc5 but lacked a putative homologue of Orc6. By expressing HsORC subunits in insect cells using the baculovirus system, we were able to identify a complex containing all six subunits. To explore the subunit-subunit interactions that are required for the assembly of HsORC, we carried out extensive co-immunoprecipitation experiments with recombinant ORC subunits expressed in different combinations. These studies revealed the following binary interactions: HsOrc2-HsOrc3, HsOrc2-HsOrc4, HsOrc3-HsOrc4, HsOrc2-HsOrc6, and HsOrc3-HsOrc6. HsOrc5 did not form stable binary complexes with any other HsORC subunit but interacted with sub-complexes containing any two of subunits HsOrc2, HsOrc3, or HsOrc4. Complex formation by HsOrc1 required the presence of HsOrc2, HsOrc3, HsOrc4, and HsOrc5 subunits. These results suggest that the subunits HsOrc2, HsOrc3, and HsOrc4 form a core upon which the ordered assembly of HsOrc5 and HsOrc1 takes place. The characterization of HsORC should facilitate the identification of human origins of DNA replication.

Assembly of functionally active Drosophila origin recognition complex from recombinant proteins

In eukaryotes the sites for the initiation of chromosomal DNA replication are believed to be determined in part by the binding of a heteromeric origin recognition complex (ORC) to DNA. We have cloned the genes encoding the subunits of the Drosophila ORC. Each of the genes is unique and can be mapped to discrete chromosomal locations implying that the pattern and developmental regulation of origin usage in Drosophila is not regulated solely by a large family of different ORC proteins. The six-subunit ORC can be reconstituted with recombinant proteins into a complex that restores DNA replication in ORC-depleted Drosophila or Xenopus egg extracts.

Role for a Xenopus Orc2-related protein in controlling DNA replication.

The six-subunit origin recognition complex (ORC) is essential for the initiation of DNA replication at specific origins in the budding yeast Saccharomyces cerevisiae. An important issue is whether DNA replication in higher eukaryotes, in which the characteristics of replication origins are poorly defined, occurs by an ORC-dependent mechanism. We have identified a Xenopus laevis Orc2-related protein (XORC2) by its ability to rescue a mitotic-catastrophe mutant of the fission yeast Schizosaccharomyces pombe. We show that immunodepletion of XORC2 from Xenopus egg extracts abolishes the replication of chromosomal DNA but not elongation synthesis on a single-stranded DNA template. Indirect immunofluorescence indicates that XORC2 binds to chromatin well before the commencement of DNA synthesis, and even under conditions that prevent the association of replication licensing factor(s) with the DNA. These findings suggest that Orc2 plays an important role at an early step of chromosomal replication in animal cells.