Human NOC3 is essential for DNA replication licensing in human cells

Noc3p (Nucleolar Complex-associated protein) is an essential protein in budding yeast DNA replication licensing. Noc3p mediates the loading of Cdc6p and MCM proteins onto replication origins during the M-to-G₁ transition by interacting with ORC (Origin Recognition Complex) and MCM (Minichromosome Maintenance) proteins. FAD24 (Factor for Adipocyte Differentiation, clone number 24), the human homolog of Noc3p (hNOC3), was previously reported to play roles in the regulation of DNA replication and proliferation in human cells. However, the role of hNOC3 in replication licensing was unclear. Here we report that hNOC3 physically interacts with multiple human pre-replicative complex (pre-RC) proteins and associates with known replication origins throughout the cell cycle. Moreover, knockdown of hNOC3 in HeLa cells abrogates the chromatin association of other pre-RC proteins including hCDC6 and hMCM, leading to DNA replication defects and eventual apoptosis in an abortive S-phase. In comparison, specific inhibition of the ribosome biogenesis pathway by preventing pre-rRNA synthesis, does not lead to any cell cycle or DNA replication defect or apoptosis in the same timeframe as the hNOC3 knockdown experiments. Our findings strongly suggest that hNOC3 plays an essential role in pre-RC formation and the initiation of DNA replication independent of its potential role in ribosome biogenesis in human cells.     

Cyclin A-dependent kinase activity affects chromatin binding of ORC, Cdc6, and MCM in egg extracts of Xenopus laevis.

The initiation of DNA replication in eukaryotes requires the loading of the origin recognition complex (ORC), Cdc6, and minichromosome maintenance (MCM) proteins onto chromatin to form the preinitiation complex. In Xenopus egg extract, the proteins Orc1, Orc2, Cdc6, and Mcm4 are underphosphorylated in interphase and hyperphosphorylated in metaphase extract. We find that chromatin binding of ORC, Cdc6, and MCM proteins does not require cyclin-dependent kinase activities. High cyclin A-dependent kinase activity inhibits the binding and promotes the release of Xenopus ORC, Cdc6, and MCM from sperm chromatin, but has no effect on chromatin binding of control proteins. Cyclin A together with ORC, Cdc6 and MCM proteins is bound to sperm chromatin in DNA replicating pseudonuclei. In contrast, high cyclin E/cdk2 was not detected on chromatin, but was found soluble in the nucleoplasm. High cyclin E kinase activity allows the binding of Xenopus ORC and Cdc6, but not MCM, to sperm chromatin, even though the kinase does not phosphorylate MCM directly. We conclude that chromatin-bound cyclin A kinase controls DNA replication by protein phosphorylation and chromatin release of Cdc6 and MCM, whereas soluble cyclin E kinase prevents rereplication during the cell cycle by the inhibition of premature MCM chromatin association.     

Identification of a Preinitiation Step in DNA Replication That Is Independent of Origin Recognition Complex and cdc6, but Dependent on cdk2

Before initiation of DNA replication, origin recognition complex (ORC) proteins, cdc6, and minichromosome maintenance (MCM) proteins bind to chromatin sequentially and form preinitiation complexes. Using Xenopus laevis egg extracts, we find that after the formation of these complexes and before initiation of DNA replication, cdc6 is rapidly removed from chromatin, possibly degraded by a cdk2-activated, ubiquitin-dependent proteolytic pathway. If this displacement is inhibited, DNA replication fails to initiate. We also find that after assembly of MCM proteins into preinitiation complexes, removal of the ORC from DNA does not block the subsequent initiation of replication. Importantly, under conditions in which both ORC and cdc6 protein are absent from preinitiation complexes, DNA replication is still dependent on cdk2 activity. Therefore, the final steps in the process leading to initiation of DNA replication during S phase of the cell cycle are independent of ORC and cdc6 proteins, but dependent on cdk2 activity.     

Schizosaccharomyces pombe Noc3 is essential for ribosome biogenesis and cell division but not DNA replication

The initiation of eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at chromosomal origins of DNA replication. Pre-RC assembly requires the essential DNA replication proteins ORC, Cdc6, and Cdt1 to load the MCM DNA helicase onto chromatin. Saccharomyces cerevisiae Noc3 (ScNoc3), an evolutionarily conserved protein originally implicated in 60S ribosomal subunit trafficking, has been proposed to be an essential regulator of DNA replication that plays a direct role during pre-RC formation in budding yeast. We have cloned Schizosaccharomyces pombe noc3(+) (Spnoc3(+)), the S. pombe homolog of the budding yeast ScNOC3 gene, and functionally characterized the requirement for the SpNoc3 protein during ribosome biogenesis, cell cycle progression, and DNA replication in fission yeast. We showed that fission yeast SpNoc3 is a functional homolog of budding yeast ScNoc3 that is essential for cell viability and ribosome biogenesis. We also showed that SpNoc3 is required for the normal completion of cell division in fission yeast. However, in contrast to the proposal that ScNoc3 plays an essential role during DNA replication in budding yeast, we demonstrated that fission yeast cells do enter and complete S phase in the absence of SpNoc3, suggesting that SpNoc3 is not essential for DNA replication in fission yeast.     

ORC, MCM, and histone hyperacetylation at the Kaposi's sarcoma-associated herpesvirus latent replication origin

The viral genome of Kaposi's sarcoma-associated herpesvirus (KSHV) persists as an extrachromosomal plasmid in latently infected cells. The KSHV latency-associated nuclear antigen (LANA) stimulates plasmid maintenance and DNA replication by binding to an approximately 150-bp region within the viral terminal repeats (TR). We have used chromatin immunoprecipitation assays to demonstrate that LANA binds specifically to the replication origin sequence within the KSHV TR in latently infected cells. The latent replication origin within the TR was also bound by LANA-associated proteins CBP, double-bromodomain-containing protein 2 (BRD2), and the origin recognition complex 2 protein (ORC2) and was enriched in hyperacetylated histones H3 and H4 relative to other regions of the latent genome. Cell cycle analysis indicated that the minichromosome maintenance complex protein, MCM3, bound TR in late-G(1)/S-arrested cells, which coincided with the loss of histone H3 K4 methylation. Micrococcal nuclease studies revealed that TRs are embedded in a highly ordered nucleosome array that becomes disorganized in late G(1)/S phase. ORC binding to TR was LANA dependent when reconstituted in transfected plasmids. DNA affinity purification confirmed that LANA, CBP, BRD2, and ORC2 bound TR specifically and identified the histone acetyltransferase HBO1 (histone acetyltransferase binding to ORC1) as a potential TR binding protein. Disruption of ORC2, MCM5, and HBO1 expression by small interfering RNA reduced LANA-dependent DNA replication of TR-containing plasmids. These findings are the first demonstration that cellular replication and origin licensing factors are required for KSHV latent cycle replication. These results also suggest that the KSHV latent origin of replication is a unique chromatin environment containing histone H3 hyperacetylation within heterochromatic tandem repeats.     

Site-specific loading of an MCM protein complex in a DNA replication initiation zone upstream of the c-MYC gene in the HeLa cell cycle

The MCM proteins participate in an orderly association, beginning with the origin recognition complex, that culminates in the initiation of chromosomal DNA replication. Among these, MCM proteins 4, 6, and 7 constitute a subcomplex that reportedly possesses DNA helicase activity. Little is known about DNA sequences initially bound by these MCM proteins or about their cell cycle distribution in the chromatin. We have determined the locations of certain MCM and associated proteins by chromatin immunoprecipitation (ChIP) in a zone of initiation of DNA replication upstream of the c-MYC gene in the HeLa cell cycle. MCM7 and its clamp-loading partner Cdc6 are highly specifically colocalized by ChIP and re-ChIP in G(1) and early S on a 198-bp segment located near the center of the initiation zone. ChIP and Re-ChIP colocalizes MCM7 and ORC1 to the same segment specifically in late G(1). MCM proteins 6 and 7 can be coimmunoprecipitated throughout the cell cycle, whereas MCM4 is reduced in the complex in late S and G(2), reappearing upon mitosis. MCM7 is not visualized by immunohistochemistry on metaphase chromosomes. MCM7 is recruited to multiple sites in chromatin in S and G(2), at which time it is not detected with ORC1. The rate of dissemination is surprisingly slow and is unlikely to be simply attributed to progression with replication forks. Results indicate sequence-specific loading of MCM proteins onto DNA in late G(1) followed by a recruitment to multiple sites in chromatin subsequent to replication.     

Interaction and assembly of murine pre-replicative complex proteins in yeast and mouse cells

Eukaryotic cells coordinate chromosome duplication by the assembly of protein complexes at origins of DNA replication by sequential binding of member proteins of the origin recognition complex (ORC), CDC6, and minichromosome maintenance (MCM) proteins. These pre-replicative complexes (pre-RCs) are activated by cyclin-dependent kinases and DBF4/CDC7 kinase. Here, we carried out a comprehensive yeast two-hybrid screen to establish sequential interactions between two individual proteins of the mouse pre-RC that are probably required for the initiation of DNA replication. The studies revealed multiple interactions among ORC subunits and MCM proteins as well as interactions between individual ORC and MCM proteins. In particular CDC6 was found to bind strongly to ORC1 and ORC2, and to MCM7 proteins. DBF4 interacts with the subunits of ORC as well as with MCM proteins. It was also demonstrated that CDC7 binds to different ORC and MCM proteins. CDC45 interacts with ORC1 and ORC6, and weakly with MCM3, -6, and -7. The three subunits of the single-stranded DNA binding protein RPA show interactions with various ORC subunits as well as with several MCM proteins. The data obtained by yeast two-hybrid analysis were paradigmatically confirmed in synchronized murine FM3A cells by immunoprecipitation of the interacting partners. Some of the interactions were found to be cell-cycle-dependent; however, most of them were cell-cycle-independent. Altogether, 90 protein-protein interactions were detected in this study, 52 of them were found for the first time in any eukaryotic pre-RC. These data may help to understand the complex interplay of the components of the mouse pre-RC and should allow us to refine its structural architecture as well as its assembly in real time.     

The MCM3 acetylase MCM3AP inhibits initiation,but not elongation,of DNA replication via interaction with MCM3

Minichromosome maintenance (MCM) proteins are essential components of pre-replication complexes, which limit DNA replication to once per cell cycle. MCM3 acetylating protein, MCM3AP, binds and acetylates MCM3 and inhibits cell cycle progression. In the present study, we examined inhibition of the cell cycle by MCM3AP in a cell-free system. We show here that wild type MCM3AP, but not the acetylase-deficient mutant, inhibits initiation of DNA replication, but not elongation. Both wild type and acetylase-deficient mutant MCM3AP, however, can bind to chromatin through interaction with MCM3. These results indicate that MCM3 acetylase activity of MCM3AP is required to inhibit initiation of DNA replication and that association of MCM3AP to chromatin alone is not sufficient for the inhibition. We also show that interaction between MCM3 and MCM3AP is essential for nuclear localization and chromatin binding of MCM3AP. Furthermore, the chromatin binding of MCM3AP is temporally correlated with that of endogenous MCM3 when cells were released from mitosis. Hence, MCM3AP is a potent natural inhibitor of the initiation of DNA replication whose action is mediated by interaction with MCM3.     

Identification of novel factors involved in or regulating initiation of DNA replication by a genome-wide phenotypic screen in Saccharomyces cerevisiae

DNA replication in eukaryotic cells is tightly regulated to ensure faithful inheritance of the genetic material. While the replicators, replication origins and many replication-initiation proteins in Saccharomyces cerevisiae have been identified and extensively studied, the detailed mechanism that controls the initiation of DNA replication is still not well understood. It is likely that some factors involved in or regulating the initiation of DNA replication have not been discovered. To identify novel DNA replication-initiation proteins and their regulators, we developed a sensitive and comprehensive phenotypic screen by combining several established genetic strategies including plasmid loss assays with plasmids containing a single versus multiple replication origins and colony color sectoring assays. We isolated dozen of mutants in previously known initiation proteins and identified several novel factors, including Ctf1p Ctf3p, Ctf4p, Ctf18p, Adk1p and Cdc60p, whose mutants lose plasmid containing a single replication origin at high rates but lose plasmid carrying multiple replication origins at lower rates. We also show that overexpression of replication initiation proteins causes synthetic dosage lethality or growth defects in ctf1 and ctf18 mutants and that Ctf1p and Ctf18p physically interact with ORC, Cdt1p and MCM proteins. Furthermore, depletion of both Ctf1p and Ctf18p prevents S phase entry, retards S phase progression, and reduces pre-RC formation during the M-to-G1 transition. These data suggest that Ctf1p and Ctf18p together play important roles in regulating the initiation of DNA replication.     

Interaction of Xenopus Cdc2 x cyclin A1 with the origin recognition complex

The initiation of DNA replication in eukaryotes is regulated in a minimum of at least two ways. First, several proteins, including origin recognition complex (ORC), Cdc6 protein, and the minichromosome maintenance (MCM) protein complex, need to be assembled on chromatin before initiation. Second, cyclin-dependent kinases regulate DNA replication in both a positive and a negative way by inducing the initiation of DNA replication at G(1)/S transition and preventing further rounds of origin firing within the same cell cycle. Here we characterize a link between the two levels. Immunoprecipitation of Xenopus origin recognition complex with anti-XOrc1 or anti-XOrc2 antibodies specifically co-immunoprecipitates a histone H1 kinase activity. The kinase activity is sensitive to several inhibitors of cyclin-dependent kinases including 6-dimethylaminopurine (6-DMAP), olomoucine, and p21(Cip1). This kinase activity also copurifies with ORC over several fractionation steps and was identified as a complex of the Cdc2 catalytic subunit and cyclin A1. Neither Cdk2 nor cyclin E could be detected in ORC immunoprecipitations. Reciprocal immunoprecipitations with anti-Xenopus Cdc2 or anti-Xenopus cyclin A1 antibodies specifically co-precipitate XOrc1 and XOrc2. Our results indicate that Xenopus ORC and Cdc2 x cyclin A1 physically interact and demonstrate a physical link between an active cyclin-dependent kinase and proteins involved in the initiation of DNA replication.     

Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis

Evidence obtained from studies with yeast and Xenopus indicate that the initiation of DNA replication is a multistep process. The origin recognition complex (ORC), Cdc6p, and minichromosome maintenance (MCM) proteins are required for establishing prereplication complexes, upon which initiation is triggered by the activation of cyclin-dependent kinases and the Dbf4p-dependent kinase Cdc7p. The identification of human homologues of these replication proteins allows investigation of S-phase regulation in mammalian cells. Using centrifugal elutriation of several human cell lines, we demonstrate that whereas human Orc2 (hOrc2p) and hMcm proteins are present throughout the cell cycle, hCdc6p levels vary, being very low in early G(1) and accumulating until cells enter mitosis. hCdc6p can be polyubiquitinated in vivo, and it is stabilized by proteasome inhibitors. Similar to the case for hOrc2p, a significant fraction of hCdc6p is present on chromatin throughout the cell cycle, whereas hMcm proteins alternate between soluble and chromatin-bound forms. Loading of hMcm proteins onto chromatin occurs in late mitosis concomitant with the destruction of cyclin B, indicating that the mitotic kinase activity inhibits prereplication complex formation in human cells.     

The Rix1 (Ipi1p-2p-3p) complex is a critical determinant of DNA replication licensing independent of their roles in ribosome biogenesis

Several replication-initiation proteins are assembled stepwise onto replicators to form pre-replicative complexes (pre-RCs) to license eukaryotic DNA replication. We performed a yeast functional proteomic screen and identified the Rix1 complex members (Ipi1p-Ipi2p/Rix1-Ipi3p) as pre-RC components and critical determinants of replication licensing and replication-initiation frequency. Ipi3p interacts with pre-RC proteins, binds chromatin predominantly at ARS sequences in a cell cycle-regulated and ORC- and Noc3p-dependent manner and is required for loading Cdc6p, Cdt1p and MCM onto chromatin to form pre-RC during the M-to-G₁ transition and for pre-RC maintenance in G₁ phase-independent of its role in ribosome biogenesis. Moreover, Ipi1p and Ipi2p, but not other ribosome biogenesis proteins Rea1p and Utp1p, are also required for pre-RC formation and maintenance, and Ipi1p, -2p and -3p are interdependent for their chromatin association and function in pre-RC formation. These results establish a new framework for the hierarchy of pre-RC proteins, where the Ipi1p-2p-3p complex provides a critical link between ORC-Noc3p and Cdc6p-Cdt1p-MCM in replication licensing.     

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.     

The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication

The initiation of eukaryotic DNA replication involves origin recruitment and activation of the MCM2-7 complex, the putative replicative helicase. Mini-chromosome maintenance (MCM)2-7 recruitment to origins in G1 requires origin recognition complex (ORC), Cdt1, and Cdc6, and activation at G1/S requires MCM10 and the protein kinases Cdc7 and S-Cdk, which together recruit Cdc45, a putative MCM2-7 cofactor required for origin unwinding. Here, we show that the Xenopus BRCA1 COOH terminus repeat-containing Xmus101 protein is required for loading of Cdc45 onto the origin. Xmus101 chromatin association is dependent on ORC, and independent of S-Cdk and MCM2-7. These results define a new factor that is required for Cdc45 loading. Additionally, these findings indicate that the initiation complex assembly pathway bifurcates early, after ORC association with the origin, and that two parallel pathways, one controlled by MCM2-7, and the other by Xmus101, cooperate to load Cdc45 onto the origin.     

The origin recognition complex marks a replication origin in the human TOP1 gene promoter

The locations of the origin recognition complex (ORC) in mammalian genomes have been elusive. We have therefore analyzed the DNA sequences associated with human ORC via in vivo cross-linking and chromatin immunoprecipitation. Antibodies specific for hOrc2 protein precipitate chromatin fragments that also contain other ORC proteins, suggesting that the proteins form multisubunit complexes on chromatin in vivo. A binding region for ORC was identified at the CpG island upstream of the human TOP1 gene. Nascent strand abundance assays show that the ORC binding region coincides with an origin of bidirectional replication. The TOP1 gene includes two well characterized matrix attachment regions. The matrix attachment region elements analyzed contain no ORC and constitute no sites for replication initiation. In initial attempts to use the chromatin immunoprecipitation technique for the identification of additional ORC sites in the human genome, we isolated a sequence close to another actively transcribed gene (TOM1) and an alphoid satellite sequence that underlies centromeric heterochromatin. Nascent strand abundance assays gave no indication that the heterochromatin sequence serves as a replication initiation site, suggesting that an ORC on this site may perform functions other than replication initiation.     

Interactions and subcellular distribution of DNA replication initiation proteins in eukaryotic cells

For initiation of eukaryotic DNA replication the origin recognition complex (ORC) associates with chromatin sites and constitutes a landing pad allowing Cdc6, Cdt1 and MCM proteins to accomplish the pre-replication complex (pre-RC). In S phase, the putative MCM helicase is assumed to move away from the ORC to trigger DNA unwinding. By using the fluorescence-based assays bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC) we show in live mammalian cells that one key interaction in pre-RC assembly, the interaction between Orc2 and Orc3, is not restricted to the nucleus but also occurs in the cytoplasm. BRET assays also revealed a direct interaction between Orc2 and nuclear localization signal (NLS)-depleted Orc3. Further, we assessed the subcellular distribution of Orc2 and Orc3 in relation to MCM proteins Mcm3 and Mcm6 as well as to a key protein involved in elongation of DNA replication, proliferating nuclear cell antigen (PCNA). Our findings illustrate the spatial complexity of the elaborated process of DNA replication as well as that the BRET and BiFC techniques are novel tools that could contribute to our understanding of the processes at the very beginning of the duplication of the genome.     

Interactions of MCP1 with components of the replication machinery in mammalian cells

Eukaryotic DNA replication starts with the assembly of a pre-replication complex (pre-RC) at replication origins. We have previously demonstrated that Metaphase Chromosome Protein 1 (MCP1) is involved in the early events of DNA replication. Here we show that MCP1 associates with proteins that are required for the establishment of the pre-replication complex. Reciprocal immunoprecipitation analysis showed that MCP1 interacted with Cdc6, ORC2, ORC4, MCM2, MCM3 and MCM7, with Cdc45 and PCNA. Immunofluorescence studies demonstrated the co-localization of MCP1 with some of those proteins. Moreover, biochemical studies utilizing chromatin-immunoprecipitation (ChIP) revealed that MCP1 preferentially binds replication initiation sites in human cells. Interestingly, although members of the pre-RC are known to interact with some hallmarks of heterochromatin, our co-immunoprecipitation and immunofluorescence analyses showed that MCP1 did not interact and did not co-localize with heterochromatic proteins including HP1β and MetH3K9. These observations suggest that MCP1 is associated with replication factors required for the initiation of DNA replication and binds to the initiation sites in loci that replicate early in S-phase. In addition, immunological assays revealed the association of MCP1 forms with histone H1 variants and mass spectrometry analysis confirmed that MCP1 peptides share common sequences with H1.2 and H1.5 subtypes.     

MCM8 is an MCM2-7-related protein that functions as a DNA helicase during replication elongation and not initiation

MCM2-7 proteins are replication factors required to initiate DNA synthesis and are currently the best candidates for replicative helicases. We show that the MCM2-7-related protein MCM8 is required to efficiently replicate chromosomal DNA in Xenopus egg extracts. MCM8 does not associate with the soluble MCM2-7 complex and binds chromatin upon initiation of DNA synthesis. MCM8 depletion does not affect replication licensing or MCM3 loading but slows down DNA synthesis and reduces chromatin recruitment of RPA34 and DNA polymerase-alpha. Recombinant MCM8 displays both DNA helicase and ATPase activities in vitro. Reconstitution experiments show that ATP binding in MCM8 is required to rescue DNA synthesis in MCM8-depleted extracts. MCM8 colocalizes with replication foci and RPA34 on chromatin. We suggest that MCM8 functions in the elongation step of DNA replication as a helicase that facilitates the recruitment of RPA34 and stimulates the processivity of DNA polymerases at replication foci.     

Yph1p,an ORC-interacting protein: potential links between cell proliferation control,DNA replication,and ribosome biogenesis

Immunoprecipitation of the origin recognition complex (ORC) from yeast extracts identified Yph1p, an essential protein containing a BRCT domain. Two Yph1p complexes were characterized. Besides ORC, MCM proteins, cell-cycle regulatory proteins, checkpoint proteins, 60S ribosomal proteins, and preribosome particle proteins were found to be associated with Yph1p. Yph1p is predominantly nucleolar and is required for 60S ribosomal subunit biogenesis and possibly for translation on polysomes. Proliferating cells depleted of Yph1p arrest in G(1) or G(2), with no cells in S phase, or significantly delay S phase progression after release from a hydroxyurea arrest. Yph1p levels decline as cells commit to exit the cell cycle, and levels vary depending on energy source. Yph1p may link cell proliferation control to DNA replication, ribosome biogenesis, and translation on polysomes.     

A WD-repeat protein stabilizes ORC binding to chromatin

Origin recognition complex (ORC) plays critical roles in the initiation of DNA replication and cell-cycle progression. In metazoans, ORC associates with origin DNA during G1 and with heterochromatin in postreplicated cells. However, what regulates the binding of ORC to chromatin is not understood. We have identified a highly conserved, leucine-rich repeats and WD40 repeat domain-containing protein 1 (LRWD1) or ORC-associated (ORCA) in human cells that interacts with ORC and modulates chromatin association of ORC. ORCA colocalizes with ORC and shows similar cell-cycle dynamics. We demonstrate that ORCA efficiently recruits ORC to chromatin. Depletion of ORCA in human primary cells and embryonic stem cells results in loss of ORC association to chromatin, concomitant reduction of MCM binding, and a subsequent accumulation in G1 phase. Our results suggest ORCA-mediated association of ORC to chromatin is critical to initiate preRC assembly in G1 and chromatin organization in post-G1 cells.