Meta-Biol

• Pathways

DNA replication pre-initiation in eukaryotic cells begins with the formation of the pre-replicative complex (pre-RC) during the late M phase and continues in the G1 phase of the mitotic cell cycle, a process also called DNA replication origin licensing. The association of initiation proteins (ORC, Cdc6, Cdt1, Mcm2-7) with the origin of replication in both S. cerevisiae and humans has been demonstrated by chromatin immunoprecipitation experiments. In S. cerevisiae, pre-replicative complexes are assembled from late M to G1. In mammalian cells as well, pre-replicative complexes are assembled from late M to G1, as shown by biochemical fractionation and immunostaining. There are significant sequence similarities among some of the proteins in the pre-replicative complex. The ORC subunits Orc1, Orc4 and Orc5 are homologous to one another and to Cdc6. The six subunits of the Mcm2-7 complex are homologous to one another. In addition, Orc1, Orc4, Orc5, Cdc6, and the Mcm2-7 subunits, are members of the AAA+ superfamily of ATPases. Since the initial identification of these pre-RC components other factors that participate in this complex have been found, including Cdt1 in human, Xenopus, S. pombe, and S. cerevisiae cells.

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).