Precise duplication of chromosomal DNA is required for genomic stability during replication. A process called replication licensing ensures that chromosomes are replicated only once per cell cycle. To form a pre-replicative complex on replication origins in the G phase, ORC first binds origin DNA and triggers the binding of Cdc6 and Cdt1. These two factors recruit a putative replicative helicase and the MCM2-7. The MCM2-7 complex promotes the unwinding of DNA origins, and the binding of additional factors to initiate the DNA replication in S-phase. Cdt1 is present during G1 and early S phase of the cell cycle and degraded during the late S, G2, and M phases [, ].This entry represents Cdt1, which can be divided into three regions based on sequence comparison and biochemical analyses: the N-terminal region (Cdt1_n) binds DNA in a sequence-, strand-, and conformation-independent manner; the middle winged helix fold (Cdt1_m) binds geminin to inhibit both binding of the MCM complex to origins of replication and DNA; and the C-terminal region (Cdt1_c) is essential for Cdt1 activity and directly interacts with the MCM2-7 helicase. The winged helix fold structure of Cdt1_m is similar to the structures of Cdt1_c and other archaeal homologues of the eukaryotic replication initiator, without apparent sequence similarity [, ].
Multicilin (also known as Idas) is required for multiciliate cell differentiation in diverse tissues []. Idas coordinately promotes cell cycle exit, centriole assembly, and FoxJ1 expression []. It interacts with Geminin, which inhibits the DNA replication licensing factor Cdt1 and regulates cell proliferation and differentiation []. The Idas-Geminin heterodimer binds Cdt1 less strongly than Geminin-Geminin [].
In eukaryotic replication licensing, Cdt1 plays a key role by recruiting the MCM2-7 complex onto the origin of chromosome. This entry represents the C-terminal domain of Cdt1 which is the most conserved region of Cdt1, essential for licensing and directly interacts with the MCM2-7 complex []. This region contains a WHD (winged helix domain), a typical DNA recognition motif []. The winged-helix fold is conserved throughout several other licensing factors, suggesting that they evolved from a common ancestor. In mouse Cdt1, the main role of the WH motif is to interact with MCM2-7 rather than with the DNA, as other winged-helix domains.
Geminin inhibits DNA replication by preventing the recruitment of the MCM complex to form the pre-replication complex at the origin of replication []. Geminin is degraded during the mitotic phase of the cell cycle. It has been proposed that geminin inhibits DNA replication during S, G2, and M phases and that geminin destruction at the metaphase-anaphase transition permits replication in the succeeding cell cycle []. Geminin can also inhibit the DNA replication licensing factor Cdt1 and regulates cell proliferation and differentiation []. Besides forming homodimers, Geminin can also form heterodimers with its homologue, Idas (also known as Multicilin) []. The Idas-Geminin heterodimer binds Cdt1 less strongly than Geminin-Geminin [].
In eukaryotic replication licensing, Cdt1 plays a key role by recruiting the MCM2-7 complex onto the origin of chromosome. This entry represents the C-terminal domain of Cdt1 which is the most conserved region of Cdt1, essential for licensing and directly interacts with the MCM2-7 complex []. This region contains a WHD (winged helix domain), a typical DNA recognition motif []. The winged-helix fold is conserved throughout several other licensing factors, suggesting that they evolved from a common ancestor. In mouse Cdt1, the main role of the WH motif is to interact with MCM2-7 rather than with the DNA, as other winged-helix domains.
The minichromosome maintenance (Mcm) complex is the replicative helicase in eukaryotic species, that plays essential roles in the initiation and elongation phases of DNA replication. During late M and early G(1), the Mcm complex is loaded onto chromatin to form prereplicative complex in a Cdt1-dependent manner. This entry represents the C-terminal domain of human Mcm6 which is the Cdt1 binding domain (CBD). The structure of CBD exhibits a typical winged helix fold that is generally involved in protein-nucleic acid interaction. The CBD failed to interact with DNA in experiments []. The CBD-Cdt1 interaction involves the helix-turn-helix motif of CBD [].
DNA replication in eukaryotes results from a highly coordinated interaction between proteins, often as part of protein complexes, and the DNA template. One of the key early steps leading to DNA replication is formation of the pre-replication complex, or pre-RC. The pre-RC is formed by the sequential binding of the origin recognition complex (ORC), Cdc6 and Cdt1 proteins, and the MCM complex. Activation of the pre-RC into the initiation complex (IC) is achieved via the action of S-phase kinases, eventually leading to the loading of the replication machinery.Recently, a novel replication complex, GINS (for Go, Ichi, Nii, and San; five, one, two, and three in Japanese), has been identified [, ]. The precise function of GINS is not known. However, genetic and two-hybrid interactions indicate that it mediates the loading of the enzymatic replication machinery at a step after the action of the S-phase kinases []. Furthermore, GINS may be a part of the replication machinery itself, since it is found associated with replicating DNA [, ]. Electron microscopy of GINS shows that it forms a ring-like structure [], reminiscent of the structure of PCNA [], the DNA polymerase delta replication clamp. This observation, coupled with the observed interactions for GINS, indicates that the complex may represent the replication clamp for DNA polymerase epsilon [].The GINS complex is essential for initiation of DNA replication in Xenopus egg extracts []. This 100kDa stable complex includes Sld5, Psf1, Psf2, and Psf3. Homologues of these components are found also in other eukaryotes. This superfamily represents the Psf3 component.
DNA replication in eukaryotes results from a highly coordinated interaction between proteins, often as part of protein complexes, and the DNA template. One of the key early steps leading to DNA replication is formation of the pre-replication complex, or pre-RC. The pre-RC is formed by the sequential binding of the origin recognition complex (ORC), Cdc6 and Cdt1 proteins, and the MCM complex. Activation of the pre-RC into the initiation complex (IC) is achieved via the action of S-phase kinases, eventually leading to the loading of the replication machinery.Recently, a novel replication complex, GINS (for Go, Ichi, Nii, and San; five, one, two, and three in Japanese), has been identified [, ]. The precise function of GINS is not known. However, genetic and two-hybrid interactions indicate that it mediates the loading of the enzymatic replication machinery at a step after the action of the S-phase kinases []. Furthermore, GINS may be a part of the replication machinery itself, since it is found associated with replicating DNA [, ]. Electron microscopy of GINS shows that it forms a ring-like structure [], reminiscent of the structure of PCNA [], the DNA polymerase delta replication clamp. This observation, coupled with the observed interactions for GINS, indicates that the complex may represent the replication clamp for DNA polymerase epsilon [].The GINS complex is essential for initiation of DNA replication in Xenopusegg extracts []. This 100kDa stable complex includes Sld5, Psf1, Psf2, and Psf3. Homologues of these components are found also in other eukaryotes. This family of proteins represents the Psf3 component.
DNA replication in eukaryotes results from a highly coordinated interaction between proteins, often as part of protein complexes, and the DNA template. One of the key early steps leading to DNA replication is formation of the prereplication complex, or pre-RC. The pre-RC is formed by the sequential binding of the origin recognition complex (ORC), Cdc6 and Cdt1 proteins, and the MCM complex. Activation of the pre-RC into the initiation complex (IC) is achieved via the action of S-phase kinases, eventually leading to the loading of the replication machinery.Recently, a novel replication complex, GINS (for Go, Ichi, Nii, and San; five, one, two, and three in Japanese), has been identified [, ]. The precise function of GINS is not known. However, genetic and two-hybrid interactions indicate that it mediates the loading of the enzymatic replication machinery at a step after the action of the S-phase kinases []. Furthermore, GINS may be a part of the replication machinery itself, since it is found associated with replicating DNA [, ]. Electron microscopy of GINS shows that it forms a ring-like structure [], reminiscent of the structure of PCNA [], the DNA polymerase delta replication clamp.This observation, coupled with the observed interactions for GINS, indicates that the complex may represent the replication clamp for DNA polymerase epsilon [].The GINS complex is essential for initiation of DNA replication in Xenopus egg extracts []. This 100kDa stable complex includes Sld5, Psf1, Psf2, and Psf3. Homologues of these components are found also in other eukaryotes. This family of proteins represents the Psf2 component.
DNA replication in eukaryotes results from a highly coordinated interaction between proteins, often as part of protein complexes, and the DNA template. One of the key early steps leading to DNA replication is formation of the prereplication complex, or pre-RC. The pre-RC is formed by the sequential binding of the origin recognition complex (ORC), Cdc6 and Cdt1 proteins, and the MCM complex. Activation of the pre-RC into the initiation complex (IC) is achieved via the action of S-phase kinases, eventually leading to the loading of the replication machinery.Recently, a novel replication complex, GINS (for Go, Ichi, Nii, and San; five, one, two, and three in Japanese), has been identified [, ]. The precise function of GINS is not known. However, genetic and two-hybrid interactions indicate that it mediates the loading of the enzymatic replication machinery at a step after the action of the S-phase kinases []. Furthermore, GINS may be a part of the replication machinery itself, since it is found associated with replicating DNA [, ]. Electron microscopy of GINS shows that it forms a ring-like structure [], reminiscent of the structure of PCNA [], the DNA polymerase delta replication clamp.This observation, coupled with the observed interactions for GINS, indicates that the complex may represent the replication clamp for DNA polymerase epsilon [].This family of proteins represents the PSF1 component (for partner of SLD five) of the GINS complex.
DNA replication in eukaryotes results from a highly coordinated interaction between proteins, often as part of protein complexes, and the DNA template. One of the key early steps leading to DNA replication is formation of the pre-replication complex, or pre-RC. The pre-RC is formed by the sequential binding of the origin recognition complex (ORC), Cdc6 and Cdt1 proteins, and the MCM complex. Activation of the pre-RC into the initiation complex (IC) is achieved via the action of S-phase kinases, eventually leading to the loading of the replication machinery.Recently, a novel replication complex, GINS (for Go, Ichi, Nii, and San; five, one, two, and three in Japanese), has been identified [, ]. The precise function of GINS is not known. However, genetic and two-hybrid interactions indicate that it mediates the loading of the enzymatic replication machinery at a step after the action of the S-phase kinases []. Furthermore, GINS may be a part of the replication machinery itself, since it is found associated with replicating DNA [, ]. Electron microscopy of GINS shows that it forms a ring-like structure [], reminiscent of the structure of PCNA [], the DNA polymerase delta replication clamp. This observation, coupled with the observed interactions for GINS, indicates that the complex may represent the replication clamp for DNA polymerase epsilon [].The GINS complex is essential for initiation of DNA replication in Xenopus egg extracts []. This 100kDa stable complex includes Sld5, Psf1, Psf2, and Psf3. Homologues of these components are found also in other eukaryotes []. The archaeal GINS complex contains two subunits (SSO0772/gins23 and SO1049/gins15 in Sulfolobus) that are poorly conserved homologues of the eukaryotic GINS subunits []. Only Gins23 is included in this entry.The eukaryotic GINS subunits are homologous. The four subunits of the complex consist of two domains each, termed the α-helical (A) and β-strand (B) domains. The A and B domains of Sld5/Psf1 are permuted with respect to Psf1/Psf3 [].
DNA replication in eukaryotes results from a highly coordinated interaction between proteins, often as part of protein complexes, and the DNA template. One of the key early steps leading to DNA replication is formation of the pre-replication complex, or pre-RC. The pre-RC is formed by the sequential binding of the origin recognition complex (ORC), Cdc6 and Cdt1 proteins, and the MCM complex. Activation of the pre-RC into the initiation complex (IC) is achieved via the action of S-phase kinases, eventually leading to the loading of the replication machinery.Recently, a novel replication complex, GINS (for Go, Ichi, Nii, and San; five, one, two, and three in Japanese), has been identified [, ]. The precise function of GINS is not known. However, genetic and two-hybrid interactions indicate that it mediates the loading of the enzymatic replication machinery at a step after the action of the S-phase kinases []. Furthermore, GINS may be a part of the replication machinery itself, since it is found associated with replicating DNA [, ]. Electron microscopy of GINS shows that it forms a ring-like structure [], reminiscent of the structure of PCNA [], the DNA polymerase delta replication clamp. This observation, coupled with the observed interactions for GINS, indicates that the complex may represent the replication clamp for DNA polymerase epsilon [].The GINS complex is essential for initiation of DNA replication in Xenopus egg extracts []. This 100kDa stable complex includes Sld5, Psf1, Psf2, and Psf3. Homologues of these components are found also in other eukaryotes []. The archaeal GINS complex contains two subunits (SSO0772/gins23 and SO1049/gins15 in Sulfolobus) that are poorly conserved homologues of the eukaryotic GINS subunits []. Only Gins23 is included in this entry.The eukaryotic GINS subunits are homologous. The four subunits of the complex consist of two domains each, termed the α-helical (A) and β-strand (B) domains. The A and B domains of Sld5/Psf1 are permuted with respect to Psf1/Psf3 [].
The Origin Recognition Complex (ORC) is a six-subunit ATP-dependent DNA-binding complex encoded in yeast by ORC1-6 []. ORC is a central component for eukaryotic DNA replication, and binds chromatin at replication origins throughout the cell cycle []. ORC directs DNA replication throughout the genome and is required for its initiation [, , ]. ORC bound at replication origins serves as the foundation for assembly of the pre-replicative complex (pre-RC), which includes Cdc6, Tah11 (aka Cdt1), and the Mcm2-7 complex [, , ]. Pre-RC assembly during G1 is required for replication licensing of chromosomes prior to DNA synthesis during S phase [, , ]. Cell cycle-regulated phosphorylation of ORC2, ORC6, Cdc6, and MCM by the cyclin-dependent protein kinase Cdc28 regulates initiation of DNA replication, including blocking reinitiation in G2/M phase [, , , ]. In yeast, ORC also plays a role in the establishment of silencing at the mating-type loci Hidden MAT Left (HML) and Hidden MAT Right (HMR) [, , ]. ORC participates in the assembly of transcriptionally silent chromatin at HML and HMR by recruiting the Sir1 silencing protein to the HML and HMR silencers [, , ]. Both ORC1 and ORC5 bind ATP, although only ORC1 has ATPase activity []. The binding of ATP by ORC1 is required for ORC binding to DNA and is essential for cell viability []. The ATPase activity of ORC1 is involved in formation of the pre-RC [, , ]. ATP binding by ORC5 is crucial for the stability of ORC as a whole. Only the ORC1-5 subunits are required for origin binding; ORC6 is essential for maintenance of pre-RCs once formed []. Interactions within ORC suggest that ORC2-3-6 may form a core complex []. ORC homologues have been found in various eukaryotes, including fission yeast, insects, amphibians, and humans []. This entry represents subunit 6, which directs DNA replication by binding to replication origins and is also involved in transcriptional silencing; interacts with Spp1 and with trimethylated histone H3; phosphorylated by Cdc28 [, ]. In Saccharomyces cerevisiae (Baker's yeast), both ends of the Orc6 interact with Cdt1 []and the N terminus mediates an interaction with the S-phase cyclin Clb5 [].
The Origin Recognition Complex (ORC) is a six-subunit ATP-dependent DNA-binding complex encoded in yeast by ORC1-6 []. ORC is a central component for eukaryotic DNA replication, and binds chromatin at replication origins throughout the cell cycle []. ORC directs DNA replication throughout the genome and is required for its initiation [, , ]. ORC bound at replication origins serves as the foundation for assembly of the pre-replicative complex (pre-RC), which includes Cdc6, Tah11 (aka Cdt1), and the Mcm2-7 complex [, , ]. Pre-RC assembly during G1 is required for replication licensing of chromosomes prior to DNA synthesis during S phase [, , ]. Cell cycle-regulated phosphorylation of ORC2, ORC6, Cdc6, and MCM by the cyclin-dependent protein kinase Cdc28 regulates initiation of DNA replication, including blocking reinitiation in G2/M phase [, , , ]. In yeast, ORC also plays a role in the establishment of silencing at the mating-type loci Hidden MAT Left (HML) and Hidden MAT Right (HMR) [, , ]. ORC participates in the assembly of transcriptionally silent chromatin at HML and HMR by recruiting the Sir1 silencing protein to the HML and HMR silencers [, , ]. Both ORC1 and ORC5 bind ATP, although only ORC1 has ATPase activity []. The binding of ATP by ORC1 is required for ORC binding to DNA and is essential for cell viability []. The ATPase activity of ORC1 is involved in formation of the pre-RC [, , ]. ATP binding by ORC5 is crucial for the stability of ORC as a whole. Only the ORC1-5 subunits are required for origin binding; ORC6 is essential for maintenance of pre-RCs once formed []. Interactions within ORC suggest that ORC2-3-6 may form a core complex []. ORC homologues have been found in various eukaryotes, including fission yeast, insects, amphibians, and humans []. This entry represents subunit 6, which directs DNA replication by binding to replication origins and is also involved in transcriptional silencing; interacts with Spp1 and with trimethylated histone H3; phosphorylated by Cdc28 [, ]. In Saccharomyces cerevisiae (Baker's yeast), both ends of the Orc6 interact with Cdt1 []and the N terminus mediates an interaction with the S-phase cyclin Clb5 [].
Cdc6 (also known as Cell division cycle 6 or Cdc18) functions as a regulator at the early stages of DNA replication, by helping to recruit and load the Minichromosome Maintenance Complex (MCM) onto DNA and may have additional roles in the control of mitotic entry. Precise duplication of chromosomal DNA is required for genomic stability during replication. Cdc6 has an essential role in DNA replication and irregular expression of Cdc6 may lead to genomic instability. Cdc6 over-expression is observed in many cancerous lesions. DNA replication begins when an origin recognition complex (ORC) binds to a replication origin site on the chromatin. Studies indicate that Cdc6 interacts with ORC through the Orc1 subunit, and that this association increases the specificity of the ORC-origins interaction. Further studies suggest that hydrolysis of Cdc6-bound ATP promotes the association of the replication licensing factor Cdt1 with origins through an interaction with Orc6 and this in turn promotes the loading of MCM2-7 helicase onto chromatin. The MCM2-7 complex promotes the unwinding of DNA origins, and the binding of additional factors to initiate the DNA replication. S-Cdk (S-phase cyclin and cyclin-dependent kinase complex) prevents rereplication by causing the Cdc6 protein to dissociate from ORC and prevents the Cdc6 and MCM proteins from reassembling at any origin. By phosphorylating Cdc6, S-Cdk also triggers Cdc6's ubiquitination. The Cdc6 protein is composed of three domains, an N-terminal AAA+ domain with Walker A and B, and Sensor-1 and -2 motifs. The central region contains a conserved nucleotide binding/ATPase domain and is a member of the ATPase superfamily. [, , , , ].The C-terminal domain of cell division control protein 6 (CDC6) assumes a winged helix fold, with a five α-helical bundle (α15-α19) structure, backed on one side by three beta strands (β6-β8). It has been shown that this domain acts as a DNA-localisation factor, however its exact function is, as yet, unknown. Putative functions include: (1) mediation of protein-protein interactions and (2) regulation of nucleotide binding and hydrolysis. Mutagenesis studies have shown that this domain is essential for appropriate CDC6 activity [].
