Abstract

We investigated the association of human origin recognition complex (ORC) proteins hOrc1p and hOrc2p with chromatin in HeLa cells. Independent procedures including limited nuclease digestion and differential salt extraction of isolated nuclei showed that a complex containing hOrc1p and hOrc2p occurs in a nuclease-resistant compartment of chromatin and can be eluted with moderate high salt concentrations. A second fraction of hOrc2p that dissociates in vitro at low salt conditions was found to occur in a chromatin compartment characterized by its high accessibility to micrococcal nuclease. Functional differences between these two sites become apparent in HeLa cells that synchronously enter the S phase after a release from a double-thymidine block. The hOrc1p/hOrc2p-containing complexes dissociate from their chromatin sites during S phase and reassociate at the end of mitosis. In contrast, the fraction of hOrc2p in nuclease-accessible, more open chromatin remains bound during all phases of the cell cycle. We propose that the hOrc1p/hOrc2p-containing complexes are components of the human origin recognition complex. Thus, the observed cell cycle-dependent release of the hOrc1p/hOrc2p-containing complexes is in line with previous studies with Xenopus and Drosophila systems, which indicated that a change in ORC stability occurs after prereplication complex formation. This could be a powerful mechanism that prevents the rereplication of already replicated chromatin in the metazoan cell cycle.

Highlights

  • Initiation of genome replication has attracted considerable attention in recent years, because it is an essential and tightly regulated process within the eukaryotic cell cycle

  • Even though the overall mechanism of prereplication complex formation is probably conserved among eukaryotes, interesting differences appear to exist between yeast and metazoan origin recognition complex (ORC) as yeast ORCs remain stably bound to origins during all cell cycle phases whereas the interactions of Xenopus and Drosophila ORCs with their cognate chromatin sites seem to vary during the cell cycle

  • As immunoprecipitations are important for the experiments to be reported below, we demonstrate in Fig. 1B that the hOrc1p-specific antibodies efficiently precipitated hOrc1p just as the human Orc2p (hOrc2p)-specific antibodies precipitated hOrc2p from crude nuclear extracts

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Summary

Introduction

Initiation of genome replication has attracted considerable attention in recent years, because it is an essential and tightly regulated process within the eukaryotic cell cycle. We investigated the association of human origin recognition complex (ORC) proteins hOrc1p and hOrc2p with chromatin in HeLa cells. The fraction of hOrc2p in nuclease-accessible, more open chromatin remains bound during all phases of the cell cycle.

Results
Conclusion

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