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Keywords:

  • replication initiation;
  • Cdk2;
  • Cdc6;
  • RPA70;
  • re-ChIP;
  • immunogold electron microscopy

Abstract

Minichromosome maintenance (MCM) proteins are essential for DNA replication in eukaryotes. A subcomplex of the MCM2-7 family members, initially characterized in yeast, is thought to serve as a eukaryotic DNA replicative helicase. MCM8 is a new family member, not present in yeast, which may function alone or with other family members in aspects of DNA metabolism, including replication initiation and elongation. Through the use of chromatin immunoprecipitation, we find that MCM8, like MCM7, colocalizes on a specific DNA segment of the c-MYC replication initiation zone (c-MYC replicator) with Cdc6, a protein potentially involved in loading MCM proteins onto DNA. The association between MCM8 and MCM7 peaks in mid G1, at the time of assembly of the prereplication complex. The association of both MCM proteins with Cdc6, however, continues even after DNA replication is complete. We also find that MCM8 colocalizes at the c-MYC replicator with chromatin-bound Cdk2. Our data indicate that any role MCM8 may play in elongation is likely to be discontinuous, in its association with DNA, from a potential role in initiation. Using immunogold electron microscopy we show that MCM8 and MCM7 differ in spatial relation to RPA70 during S phase. Our data strongly suggest that MCM8 functions with other known replication proteins in processes which accompany DNA replication, especially initiation, and which are specifically adapted to suit higher eukaryotes. Microsc. Res. Tech, 2008. © 2007 Wiley-Liss, Inc.