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.     

MCM9 binds Cdt1 and is required for the assembly of prereplication complexes

Prereplication complexes (pre-RCs) define potential origins of DNA replication and allow the recruitment of the replicative DNA helicase MCM2-7. Here, we characterize MCM9, a member of the MCM2-8 family. We demonstrate that MCM9 binds to chromatin in an ORC-dependent manner and is required for the recruitment of the MCM2-7 helicase onto chromatin. Its depletion leads to a block in pre-RC assembly, as well as DNA replication inhibition. We show that MCM9 forms a stable complex with the licensing factor Cdt1, preventing an excess of geminin on chromatin during the licensing reaction. Our data suggest that MCM9 is an essential activating linker between Cdt1 and the MCM2-7 complex, required for loading the MCM2-7 helicase onto DNA replication origins. Thus, Cdt1, with its two opposing regulatory binding factors MCM9 and geminin, appears to be a major platform on the pre-RC to integrate cell-cycle signals.     

CpG methylation of DNA restricts prereplication complex assembly in Xenopus egg extracts

In a Xenopus egg replication system, the origin recognition complex (ORC) does not bind to CpG methylated DNA and DNA replication is inhibited. Insertion of low density CpG DNA of at least 1.2 kb into methylated plasmids rescues both replication and ORC binding. Using this pseudo-origin, we find that ORC binding is restricted to low-CpG-density DNA; however, MCM is loaded onto both weakly and highly methylated DNA and occupies at least approximately 2 kb of DNA. Replication initiates coincident with MCM, and even the most distally bound MCM is associated with sites of replication initiation. These results suggest that in metazoans MCM is loaded onto and initiates replication over a large region distant from ORC.     

MCM interference during licensing of DNA replication in Xenopus egg extracts-Possible Role of a C-terminal region of MCM3-

The minichromosome maintenance (MCM) complex, consisting of six subunits, Mcm2-7, is loaded onto replication origins through loading factors (origin recognition complex [ORC], Cdc6, and Cdt1) and forms an MCM double hexamer that licenses the initiation of DNA replication. Previous studies with Xenopus egg extracts showed that loading factors, especially Cdc6, dissociate from chromatin on MCM loading, but the molecular mechanism and physiological significance remain largely unknown. Using a cell-free system for MCM loading onto plasmid DNA in Xenopus egg extracts, we found that MCM loaded onto DNA prevents DNA binding of the loading factors ORC, Cdc6, and Cdt1. We further report that a peptide of the C-terminal region of MCM3 (MCM3-C), previously implicated in the initial association with ORC/Cdc6 in budding yeast, prevents ORC/Cdc6/Cdt1 binding to DNA in the absence of MCM loading. ATP-γ-S suppresses inhibitory activities of both the MCM loaded onto DNA and the MCM3-C peptide. Other soluble factors in the extract, but neither MCM nor Cdt1, are required for the activity. Conservation of the amino acid sequences of MCM3-C and its activity in vertebrates implies a novel negative autoregulatory mechanism that interferes with MCM loading in the vicinity of licensed origins to ensure proper origin licensing.     

Radiation-mediated proteolysis of CDT1 by CUL4-ROC1 and CSN complexes constitutes a new checkpoint

Genomic integrity is maintained by checkpoints that guard against undesired replication after DNA damage. Here, we show that CDT1, a licensing factor of the pre-replication complex (preRC), is rapidly proteolysed after UV- or gamma-irradiation. The preRC assembles on replication origins at the end of mitosis and during G1 to license DNA for replication in S phase. Once the origin recognition complex (ORC) binds to origins, CDC6 and CDT1 associate with ORC and promote loading of the MCM2-7 proteins onto chromatin, generating the preRC. We show that radiation-mediated CDT1 proteolysis is independent of ATM and CHK2 and can occur in G1-phase cells. Loss of the COP9-signalosome (CSN) or CUL4-ROC1 complexes completely suppresses CDT1 proteolysis. CDT1 is specifically polyubiquitinated by CUL4 complexes and the interaction between CDT1 and CUL4 is regulated in part by gamma-irradiation. Our study reveals an evolutionarily conserved and uncharacterized G1 checkpoint that induces CDT1 proteolysis by the CUL4-ROC1 ubiquitin E3 ligase and CSN complexes in response to DNA damage.     

Dynamic association of ORCA with prereplicative complex components regulates DNA replication initiation

In eukaryotes, initiation of DNA replication requires the assembly of a multiprotein prereplicative complex (pre-RC) at the origins. We recently reported that a WD repeat-containing protein, origin recognition complex (ORC)-associated (ORCA/LRWD1), plays a crucial role in stabilizing ORC to chromatin. Here, we find that ORCA is required for the G(1)-to-S-phase transition in human cells. In addition to binding to ORC, ORCA associates with Cdt1 and its inhibitor, geminin. Single-molecule pulldown experiments demonstrate that each molecule of ORCA can bind to one molecule of ORC, one molecule of Cdt1, and two molecules of geminin. Further, ORCA directly interacts with the N terminus of Orc2, and the stability of ORCA is dependent on its association with Orc2. ORCA associates with Orc2 throughout the cell cycle, with Cdt1 during mitosis and G(1), and with geminin in post-G(1) cells. Overexpression of geminin results in the loss of interaction between ORCA and Cdt1, suggesting that increased levels of geminin in post-G(1) cells titrate Cdt1 away from ORCA. We propose that the dynamic association of ORCA with pre-RC components modulates the assembly of its interacting partners on chromatin and facilitates DNA replication initiation.     

The ORC/Cdc6/MCM2-7 complex facilitates MCM2-7 dimerization during prereplicative complex formation

The replicative mini-chromosome-maintenance 2–7 (MCM2-7) helicase is loaded in Saccharomyces cerevisiae and other eukaryotes as a head-to-head double-hexamer around origin DNA. At first, ORC/Cdc6 recruits with the help of Cdt1 a single MCM2-7 hexamer to form an ‘initial’ ORC/Cdc6/Cdt1/MCM2-7 complex. Then, on ATP hydrolysis and Cdt1 release, the ‘initial’ complex is transformed into an ORC/Cdc6/MCM2-7 (OCM) complex. However, it remains unclear how the OCM is subsequently converted into a MCM2-7 double-hexamer. Through analysis of MCM2-7 hexamer-interface mutants we discovered a complex competent for MCM2-7 dimerization. We demonstrate that these MCM2-7 mutants arrest during prereplicative complex (pre-RC) assembly after OCM formation, but before MCM2-7 double-hexamer assembly. Remarkably, only the OCM complex, but not the ‘initial’ ORC/Cdc6/Cdt1/MCM2-7 complex, is competent for MCM2-7 dimerization. The MCM2-7 dimer, in contrast to the MCM2-7 double-hexamer, interacts with ORC/Cdc6 and is salt-sensitive, classifying the arrested complex as a helicase-loading intermediate. Accordingly, we found that overexpression of the mutants cause cell-cycle arrest and dominant lethality. Our work identifies the OCM complex as competent for MCM2-7 dimerization, reveals MCM2-7 dimerization as a limiting step during pre-RC formation and defines critical mechanisms that explain how origins are licensed.     

Genome based identification and analysis of the pre-replicative complex of Arabidopsis thaliana

Eukaryotic DNA replication requires an ordered and regulated machinery to control G1/S transition. The formation of the pre-replicative complex (pre-RC) is a key step involved in licensing DNA for replication. Here, we identify all putative components of the full pre-RC in the genome of the model plant Arabidopsis thaliana. Different from the other eukaryotes, Arabidopsis houses in its genome two putative homologs of ORC1, CDC6 and CDT1. Two mRNA variants of AtORC4 subunit, with different temporal expression patterns, were also identified. Two-hybrid binary interaction assays suggest a primary architectural organization of the Arabidopsis ORC, in which AtORC3 plays a central role in maintaining the complex associations. Expression profiles differ among pre-RC components suggesting the existence of various forms of the complex, possibly playing different roles during development. In addition, the expression of the putative pre-RC genes in non-proliferating plant tissues suggests that they might have roles in processes other than DNA replication licensing.     

How to load a replicative helicase onto chromatin: A more and more complex matter during evolution

In all eukaryotes, the heterohexameric MCM2-7 complex functions as the main replicative helicase during S phase. During early G1 phase, it is recruited onto chromatin in a sequence of reactions called pre-replication complex (pre-RC) formation or DNA licensing. This process is ATP-dependent and at least two different chromatin-bound ATPase activities are required besides several others essential, but not enzymatically active, proteins. Although functionally conserved during evolution, pre-RC formation and the way the MCM2-7 helicase is loaded onto DNA are more complex in metazoans than in single-cell eukaryotes. Recently, we characterized a new essential factor for pre-RC assembly and DNA licensing, the vertebrate-specific MCM9 protein that contains not only an ATPase but also a helicase domain. MCM9 adds another layer of complexity to how vertebrates achieve and regulate the loading of the MCM2-7 helicase and DNA replication.     

The interaction networks of the budding yeast and human DNA replication-initiation proteins

DNA replication is a stringently regulated cellular process. In proliferating cells, DNA replication-initiation proteins (RIPs) are sequentially loaded onto replication origins during the M-to-G₁ transition to form the pre-replicative complex (pre-RC), a process known as replication licensing. Subsequently, additional RIPs are recruited to form the pre-initiation complex (pre-IC). RIPs and their regulators ensure that chromosomal DNA is replicated exactly once per cell cycle. Origin recognition complex (ORC) binds to, and marks replication origins throughout the cell cycle and recruits other RIPs including Noc3p, Ipi1-3p, Cdt1p, Cdc6p and Mcm2-7p to form the pre-RC. The detailed mechanisms and regulation of the pre-RC and its exact architecture still remain unclear. In this study, pairwise protein-protein interactions among 23 budding yeast and 16 human RIPs were systematically and comprehensively examined by yeast two-hybrid analysis. This study tested 470 pairs of yeast and 196 pairs of human RIPs, from which 113 and 96 positive interactions, respectively, were identified. While many of these interactions were previously reported, some were novel, including various ORC and MCM subunit interactions, ORC self-interactions, and the interactions of IPI3 and NOC3 with several pre-RC and pre-IC proteins. Ten of the novel interactions were further confirmed by co-immunoprecipitation assays. Furthermore, we identified the conserved interaction networks between the yeast and human RIPs. This study provides a foundation and framework for further understanding the architectures, interactions and functions of the yeast and human pre-RC and pre-IC.     

Sequential ATP hydrolysis by Cdc6 and ORC directs loading of the Mcm2-7 helicase

Loading of the Mcm2-7 DNA replicative helicase onto origin-proximal DNA is a critical and tightly regulated event during the initiation of eukaryotic DNA replication. The resulting protein-DNA assembly is called the prereplicative complex (pre-RC), and its formation requires the origin recognition complex (ORC), Cdc6, Cdt1, and ATP. ATP hydrolysis by ORC is required for multiple rounds of Mcm2-7 loading. Here, we investigate the role of ATP hydrolysis by Cdc6 during pre-RC assembly. We find that Cdc6 is an ORC- and origin DNA-dependent ATPase that functions at a step preceding ATP hydrolysis by ORC. Inhibiting Cdc6 ATP hydrolysis stabilizes Cdt1 on origin DNA and prevents Mcm2-7 loading. In contrast, the initial association of Mcm2-7 with the other pre-RC components does not require ATP hydrolysis by Cdc6. Importantly, these coordinated yet distinct functions of ORC and Cdc6 ensure the correct temporal and spatial regulation of pre-RC formation.     

Pre-replicative complex assembly with purified proteins

Licensing of origins of eukaryotic DNA replication involves the loading of six minichromosome maintenance proteins (Mcm2-7) into pre-replicative complexes (pre-RCs). The assembly of the pre-RC is restricted to G1 phase of the cell cycle, which is crucial to ensure once per cell cycle DNA replication. Mcm2-7 is loaded by the action of the origin recognition complex (ORC), Cdc6 and Cdt1 and requires ATP. In vitro reconstitution of this reaction has shown that Mcm2-7 is loaded onto DNA as a symmetrical head-to-head double hexamer. We describe in detail how pre-RC proteins are purified and used to reconstitute pre-RC formation in vitro. This method is useful for studying the biochemical mechanisms of Mcm2-7 loading as well as subsequent steps in DNA replication.     

Dynamics of Pre-replicative Complex Assembly

The pre-replicative complex (pre-RC) is formed at all potential origins of replication through the action of the origin recognition complex (ORC), Cdc6, Cdt1, and the Mcm2-7 complex. The end result of pre-RC formation is the loading of the Mcm2-7 replicative helicase onto origin DNA. We examined pre-RC formation in vitro and found that it proceeds through separable binding events. Origin-bound ORC recruits Cdc6, and this ternary complex then promotes helicase loading in the presence of a pre-formed Mcm2-7-Cdt1 complex. Using a stepwise pre-RC assembly assay, we investigated the fate of pre-RC components during later stages of the reaction. We determined that helicase loading is accompanied by dissociation of ORC, Cdc6, and Cdt1 from origin DNA. This dissociation requires ATP hydrolysis at a late stage of pre-RC assembly. Our results indicate that pre-RC formation is a dynamic process.     

Regulation of germline stem cell proliferation downstream of nutrient sensing

In eukaryotic cells, replication of genomic DNA initiates from multiple replication origins distributed on multiple chromosomes. To ensure that each origin is activated precisely only once during each S phase, a system has evolved which features periodic assembly and disassembly of essential pre-replication complexes (pre-RCs) at replication origins. The pre-RC assembly reaction involves the loading of a presumptive replicative helicase, the MCM2-7 complexes, onto chromatin by the origin recognition complex (ORC) and two essential factors, CDC6 and Cdt1. The eukaryotic cell cycle is driven by the periodic activation and inactivation of cyclin-dependent kinases (Cdks) and assembly of pre-RCs can only occur during the low Cdk activity period from late mitosis through G1 phase, with inappropriate re-assembly suppressed during S, G2, and M phases. It was originally suggested that inhibition of Cdt1 function after S phase in vertebrate cells is due to geminin binding and that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance induces re-replication. However, recent progress has revealed that Cdt1 activity is more strictly regulated by two other mechanisms in addition to geminin: (1) functional and SCF^Skp2-mediated proteolytic regulation through phosphorylation by Cdks; and (2) replication-coupled proteolysis mediated by the Cullin4-DDB1^Cdt2 ubiquitin ligase and PCNA, an eukaryotic sliding clamp stimulating replicative DNA polymerases. The tight regulation implies that Cdt1 control is especially critical for the regulation of DNA replication in mammalian cells. Indeed, Cdt1 overexpression evokes chromosomal damage even without re-replication. Furthermore, deregulated Cdt1 induces chromosomal instability in normal human cells. Since Cdt1 is overexpressed in cancer cells, this could be a new molecular mechanism leading to carcinogenesis. In this review, recent insights into Cdt1 function and regulation in mammalian cells are discussed.     

Metazoan origin selection: origin recognition complex chromatin binding is regulated by CDC6 recruitment and ATP hydrolysis

Using a plasmid competition assay, we have measured the stability of origin recognition complex (ORC) associated with sperm chromatin under physiological conditions. Under conditions in which pre-RCs are formed, both ORC and CDC6 dissociate from sperm chromatin with a relatively fast t(1/2) of 15 min. ORC dissociation from chromatin is regulated through the recruitment of CDC6 and MCM proteins as well as ATP hydrolysis. The t(1/2) for ORC alone in the absence of Cdc6 is 40 min and increases 8-fold to >2 h when Cdc6 is present. Strikingly, the presence of a non-hydrolyzable ATP derivative, ATPgammaS, not only increases both ORC and CDC6 t(1/2) but also inhibits the loading of MCM. The very stable association of ORC and Cdc6 with chromatin in this sequence-independent replication system suggests that origin selection in metazoans cannot be strictly dependent on the interaction of ORCs with specific DNA binding sequences.     

The Involvement of Acidic Nucleoplasmic DNA-binding Protein (And-1) in the Regulation of Prereplicative Complex (pre-RC) Assembly in Human Cells

DNA replication in all eukaryotes starts with the process of loading the replicative helicase MCM2–7 onto chromatin during late mitosis of the cell cycle. MCM2–7 is a key component of the prereplicative complex (pre-RC), which is loaded onto chromatin by the concerted action of origin recognition complex, Cdc6, and Cdt1. Here, we demonstrate that And-1 is assembled onto chromatin in late mitosis and early G₁ phase before the assembly of pre-RC in human cells. And-1 forms complexes with MCM2–7 to facilitate the assembly of MCM2–7 onto chromatin at replication origins in late mitosis and G₁ phase. We also present data to show that depletion of And-1 significantly reduces the interaction between Cdt1 and MCM7 in G₁ phase cells. Thus, human And-1 facilitates loading of the MCM2–7 helicase onto chromatin during the assembly of pre-RC.     

Nuclear organization of DNA replication initiation proteins in mammalian cells.

Origin recognition complex (ORC), CDC6, and MCM proteins assemble sequentially to form prereplication chromatin. However, their organization remains largely unclear in mammalian cells. Here we show that ORC1 proteins are associated with non-chromatin nuclear structures and assemble in nuclear foci in mammalian cells using an in vivo chemical cross-linking method. CDC6 proteins were also found to assemble in nuclear foci on non-chromatin nuclear structures, although their physical association with ORC1 has been undetectable. In contrast to the situation in yeast cells, CDC6 was found to remain associated with non-chromatin nuclear structures even after cells entered into S phase. Instead, ORC1 proteins were found to be degraded by a proteasome-dependent pathway during S phase. We also found that some ORC2 proteins are associated with non-chromatin nuclear structures like ORC1, although the remainder binds to nuclease-sensitive chromatin. Further analyses indicate that ORC2 physically interacts with ORC1 on non-chromatin nuclear structures. On the other hand, our results suggest that although a small proportion of MCM complexes are loaded onto chromatin regions near ORC foci, most of them are more widely distributed. Possible relations between such organization of prereplication chromatin and complicated origin specification in higher eukaryotic cells are discussed.     

ATP hydrolysis by ORC catalyzes reiterative Mcm2-7 assembly at a defined origin of replication

The origin recognition complex (ORC) is a six-subunit, ATP-regulated, DNA binding protein that is required for the formation of the prereplicative complex (pre-RC), an essential replication intermediate formed at each origin of DNA replication. In this study, we investigate the mechanism of ORC function during pre-RC formation and how ATP influences this event. We demonstrate that ATP hydrolysis by ORC requires the coordinate function of the Orc1 and Orc4 subunits. Mutations that eliminate ORC ATP hydrolysis do not support cell viability and show defects in pre-RC formation. Pre-RC formation involves reiterative loading of the putative replicative helicase, Mcm2-7, at the origin. Importantly, preventing ORC ATP hydrolysis inhibits this repeated Mcm2-7 loading. Our findings indicate that ORC is part of a helicase-loading molecular machine that repeatedly assembles Mcm2-7 complexes onto origin DNA and suggest that the assembly of multiple Mcm2-7 complexes plays a critical role in origin function.     

Regulation of replication licensing by acetyltransferase Hbo1

The initiation of DNA replication is tightly regulated in eukaryotic cells to ensure that the genome is precisely duplicated once and only once per cell cycle. This is accomplished by controlling the assembly of a prereplicative complex (pre-RC) which involves the sequential binding to replication origins of the origin recognition complex (ORC), Cdc6/Cdc18, Cdt1, and the minichromosome maintenance complex (Mcm2-Mcm7, or Mcm2-7). Several mechanisms of pre-RC regulation are known, including ATP utilization, cyclin-dependent kinase levels, protein turnover, and Cdt1 binding by geminin. Histone acetylation may also affect the initiation of DNA replication, but at present neither the enzymes nor the steps involved are known. Here, we show that Hbo1, a member of the MYST histone acetyltransferase family, is a previously unrecognized positive regulatory factor for pre-RC assembly. When Hbo1 expression was inhibited in human cells, Mcm2-7 failed to associate with chromatin even though ORC and Cdc6 loading was normal. When Xenopus egg extracts were immunodepleted of Xenopus Hbo1 (XHbo1), chromatin binding of Mcm2-7 was lost, and DNA replication was abolished. The binding of Mcm2-7 to chromatin in XHbo1-depleted extracts could be restored by the addition of recombinant Cdt1.     

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.