Human Minichromosome Maintenance Proteins and Human Origin Recognition Complex 2 Protein on Chromatin

Marion Ritzi,Martina Baack,Christine Musahl,Piotr Romanowski,Ron A Laskey,Rolf Knippers

doi:10.1074/jbc.273.38.24543

Abstract

Minichromosome maintenance (Mcm) proteins and the constituents of the origin recognition complex (Orc) are essential components of the eukaryotic replication initiation apparatus. Published evidence strongly suggests that the binding of Mcm proteins to chromatin is contingent upon the prior binding of Orc proteins. Here we use two different approaches to investigate the presence of the human ORC2 protein and of Mcm proteins on chromatin of HeLa cells in various cell cycle phases. First, we mobilized chromatin-bound proteins by micrococcal nuclease and analyzed the resulting digestion products by sucrose gradient centrifugations. Under digestion conditions when Mcm proteins were almost entirely released from chromatin, ORC2 protein was found to be associated with chromatin fragments containing several hundred base pairs of DNA. Second, we used an in vivo cross-linking procedure to covalently link Mcm proteins and ORC2 to DNA by short exposure of intact HeLa cells to formaldehyde. Specific immunoprecipitations revealed that cross-linked nucleoprotein fragments carried either Mcm proteins or ORC2 protein, but not both. Based on the lengths of the DNA fragments in immunoprecipitates, we estimate that the distance between chromatin-bound ORC2 protein and chromatin-bound Mcm proteins must be at least 500-1000 base pairs in HeLa cells.

Highlights

Considerable progress has recently been made in describing the components of the machinery required for the initiation of eukaryotic genome replication
This complex has the tendency to desintegrate into more stable subcomplexes of which one is a dimer of the MCM3 protein (MCM3p) and the MCM5 protein (MCM5p), while the second is a trimer of proteins MCM4 (MCM4p), MCM6 (MCM6p), and MCM7 (MCM7p)
Immunofluorescence studies with nuclei from cultured mammalian cells had previously shown that Minichromosome maintenance (Mcm) proteins dissociate from their chromatin binding sites when cells progress through the S phase of the cell cycle [21,22,23]

Summary

Introduction

Considerable progress has recently been made in describing the components of the machinery required for the initiation of eukaryotic genome replication. The present study was performed to investigate the mode of binding of Mcm proteins and ORC2p, a member of the origin recognition complex, on human chromatin. Using specific antibodies in immunoblot experiments, we determined that Mcm proteins on chromatin decreased during S phase, but rapidly bound to chromatin again after completion of mitosis (Fig. 1B).

Results

Conclusion