ORC2

Abstract: 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.

Abstract: Although it has been demonstrated that discrete origins of DNA replication exist in eukaryotic cellular chromosomes, the detailed organization of a eukaryotic cellular origin remains to be determined. Linker substitution mutations were constructed across the entire Saccharomyces cerevisiae chromosomal origin, ARS1. Functional studies of these mutants revealed one essential element (A), which includes a match to the ARS consensus sequence, and three additional elements (B1, B2, and B3), which collectively are also essential for origin function. These four elements arranged exactly as in ARS1, but surrounded by completely unrelated sequence, functioned as an efficient origin. Element B3 is the binding site for the transcription factor-origin binding protein ABF1. Other transcription factor binding sites substitute for the B3 element and a trans-acting transcriptional activation domain is required. The multipartite nature of a chromosomal replication origin and the role of transcriptional activators in its function present a striking similarity to the organization of eukaryotic promoters.

Abstract: The genes encoding two of the subunits of the Saccharomyces cerevisiae origin recognition complex (ORC) have been isolated. Characterization of a temperature-sensitive mutation in the gene encoding the 72-kD subunit of ORC (ORC2) indicates that this protein complex functions early in the DNA replication process. Moreover, ORC derived from orc2ts cells is defective for DNA binding. Others have shown a defect in orc2ts cells in transcriptional silencing at the silent mating-type loci. Consistent with this finding, ORC specifically binds to each of the four mating-type silencers identified in yeast. These findings support the hypothesis that ORC acts as an initiator protein at yeast origins of DNA replication and suggest that ORC also functions in the determination of transcriptional domains.