Pathway: Assembly of the ORC complex at the origin of replication
Reactions in pathway: Assembly of the ORC complex at the origin of replication :
Assembly of the ORC complex at the origin of replication
Human ORC1 can associate with DNA origin of replication sites independently of other origin of replication complex (ORC) subunits (Hoshina et al. 2013; Eladl et al. 2021). ORC1 localizes to condensed chromosomes during early mitosis (M phase) and serves as a nucleating center for the assembly of the ORC and, subsequently, the pre-replication complex. ORC1 remains associated with late replication origins throughout late G1. Upon S phase entry, ORC1 undergoes ubiquitin-mediated degradation, leading to dissociation of the ORC from chromatin (Kara et al. 2015).
Most human replication origins contain guanine (G)-rich sequences which may form G-quadruplex (G4) structures (Besnard et al. 2012) and these G4 structures may mediate the recognition of replication origins by ORC1 (Hoshina et al. 2013; Eladl et al. 2021). Besides binding to nucleosome-free replication origin DNA, ORC1 interacts with neighboring nucleosomes (Hizume et al. 2013), in particular with nucleosomes containing histone H4 dimethylated at lysine 21 (H4K20me2 mark), which is enriched at replication origins. Binding of ORC1 to H4K20me2 facilitates ORC1 binding to replication origins and ORC chromatin loading (Kuo et al. 2012, Zhang et al. 2015).
ORC1 binding sites are universally associated with transcription start sites (TSSs) of coding and non-coding RNAs. Replication origins associated with moderate to high transcription level TSSs (belonging to coding RNAs) fire in early S phase, while those associated with low transcription level TSSs (belonging to non-coding RNAs) fire throughout the S phase (Dellino et al. 2013).
ORC2 forms a heterodimer with ORC3, which is a prerequisite for the association of ORC5 and, subsequently, ORC4 (Ranjan and Gossen 2006; Siddiqui and Stillman 2007). ORC1 binds to the ORC(2-5) complex in the nucleus to form a stable ORC(1-5) complex (Radichev et al. 2006; Ghosh et al. 2011). ORC1 is necessary for the association of the ORC(2-5) complex to chromatin (Radichev et al. 2006). The ORC(2-5) complex exhibits a tightly autoinhibited conformation, with the winged-helix domain (WHD) of ORC2 completely blocking the central DNA-binding channel. Binding of ORC1 remodels the WHD of ORC2, moving it away from the central channel and partially relieving the autoinhibition (Cheng et al. 2020, Jaremko et al. 2020). ORC6 associates with the ORC(1-5) complex to form the ORC(1-6) complex (Ghosh et al. 2011). The association of ORC6 with the ORC(1-5) complex is weak and it frequently does not co-immunoprecipitate with the other ORC(1-5) subunits. ORC4 is the only ORC(1-5) subunit that was shown to directly bind to ORC6 (Radichev et al. 2006). Some ORC6 mutations reported in Meier-Gorlin syndrome were shown to interfere with ORC6 incorporation into the ORC (Balasov et al. 2015).
Most human replication origins contain guanine (G)-rich sequences which may form G-quadruplex (G4) structures (Besnard et al. 2012) and these G4 structures may mediate the recognition of replication origins by ORC1 (Hoshina et al. 2013; Eladl et al. 2021). Besides binding to nucleosome-free replication origin DNA, ORC1 interacts with neighboring nucleosomes (Hizume et al. 2013), in particular with nucleosomes containing histone H4 dimethylated at lysine 21 (H4K20me2 mark), which is enriched at replication origins. Binding of ORC1 to H4K20me2 facilitates ORC1 binding to replication origins and ORC chromatin loading (Kuo et al. 2012, Zhang et al. 2015).
ORC1 binding sites are universally associated with transcription start sites (TSSs) of coding and non-coding RNAs. Replication origins associated with moderate to high transcription level TSSs (belonging to coding RNAs) fire in early S phase, while those associated with low transcription level TSSs (belonging to non-coding RNAs) fire throughout the S phase (Dellino et al. 2013).
ORC2 forms a heterodimer with ORC3, which is a prerequisite for the association of ORC5 and, subsequently, ORC4 (Ranjan and Gossen 2006; Siddiqui and Stillman 2007). ORC1 binds to the ORC(2-5) complex in the nucleus to form a stable ORC(1-5) complex (Radichev et al. 2006; Ghosh et al. 2011). ORC1 is necessary for the association of the ORC(2-5) complex to chromatin (Radichev et al. 2006). The ORC(2-5) complex exhibits a tightly autoinhibited conformation, with the winged-helix domain (WHD) of ORC2 completely blocking the central DNA-binding channel. Binding of ORC1 remodels the WHD of ORC2, moving it away from the central channel and partially relieving the autoinhibition (Cheng et al. 2020, Jaremko et al. 2020). ORC6 associates with the ORC(1-5) complex to form the ORC(1-6) complex (Ghosh et al. 2011). The association of ORC6 with the ORC(1-5) complex is weak and it frequently does not co-immunoprecipitate with the other ORC(1-5) subunits. ORC4 is the only ORC(1-5) subunit that was shown to directly bind to ORC6 (Radichev et al. 2006). Some ORC6 mutations reported in Meier-Gorlin syndrome were shown to interfere with ORC6 incorporation into the ORC (Balasov et al. 2015).
Although, DNA replication occurs in the S phase of the cell cycle, the formation of the DNA replication pre-initiation complex begins during G1 phase.
Studies in the past decade have suggested that the basic mechanism of DNA replication initiation is conserved in all kingdoms of life. Initiation in unicellular eukaryotes, in particular Saccharomyces cerevisiae (budding yeast), is well understood, and has served as a model for studies of DNA replication initiation in multicellular eukaryotes, including humans. In general terms, the first step of initiation is the binding of the replication initiator to the origin of replication. The replicative helicase is then assembled onto the origin, usually by a helicase assembly factor. Either shortly before or shortly after helicase assembly, some local unwinding of the origin of replication occurs in a region rich in adenine and thymine bases (often termed a DNA unwinding element, DUE). The unwound region provides the substrate for primer synthesis and initiation of DNA replication. The best-defined eukaryotic origins are those of S. cerevisiae, which have well-conserved sequence elements for initiator binding, DNA unwinding and binding of accessory proteins. In multicellular eukaryotes, unlike S. cerevisiae, these loci appear not to be defined by the presence of a DNA sequence motif. Indeed, choice of replication origins in a multicellular eukaryote may vary with developmental stage and tissue type. In cell-free models of metazoan DNA replication, such as the one provided by Xenopus egg extracts, there are only limited DNA sequence specificity requirements for replication initiation (Kelly & Brown 2000; Bell & Dutta 2002; Marahrens & Stillman 1992; Cimbora & Groudine 2001; Mahbubani et al 1992, Hyrien & Mechali 1993).