Site-specific Loading of an MCM Protein Complex in a DNA Replication Initiation Zone Upstream of the c-MYC Gene in the HeLa Cell Cycle

Abstract

The MCM proteins participate in an orderly association, beginning with the origin recognition complex, that culminates in the initiation of chromosomal DNA replication. Among these, MCM proteins 4, 6, and 7 constitute a subcomplex that reportedly possesses DNA helicase activity. Little is known about DNA sequences initially bound by these MCM proteins or about their cell cycle distribution in the chromatin. We have determined the locations of certain MCM and associated proteins by chromatin immunoprecipitation (ChIP) in a zone of initiation of DNA replication upstream of the c-MYC gene in the HeLa cell cycle. MCM7 and its clamploading partner Cdc6 are highly specifically colocalized by ChIP and re-ChIP in G 1 and early S on a 198-bp segment located near the center of the initiation zone. ChIP and Re-ChIP colocalizes MCM7 and ORC1 to the same segment specifically in late G 1 . MCM proteins 6 and 7 can be coimmunoprecipitated throughout the cell cycle, whereas MCM4 is reduced in the complex in late S and G 2 , reappearing upon mitosis. MCM7 is not visualized by immunohistochemistry on metaphase chromosomes. MCM7 is recruited to multiple sites in chromatin in S and G 2 , at which time it is not detected with ORC1. The rate of dissemination is surprisingly slow and is unlikely to be simply attributed to progression with replication forks. Results indicate sequence-specific loading of MCM proteins onto DNA in late G 1 followed by a recruitment to multiple sites in chromatin subsequent to replication. Members of the minichromosome maintenance (MCM)1 protein family were first characterized in Saccharomyces cerevisiae as essential for plasmid maintenance during the cell cycle (reviewed in Refs. 1-3). Each of the six proteins MCM2, MCM3, MCM4, MCM5, MCM6, and MCM7 has a counterpart in yeast, and each is highly conserved throughout eukaryotes. Deletion of genes encoding any one of these six are lethal in S. cerevisiae or Schizosaccharomyces pombe (3, 4), and these proteins have been ascribed as essential for initiation of DNA replication (5, 6). These proteins have been co-isolated as a single complex, and complexes of various combinations of these proteins have been implicated in licensing DNA for initiation of replication in Xenopus egg extracts (7,8). Another MCM protein, MCM10, is reportedly required for phosphorylation of components of the MCM2-7 complex prior to initiation (9) and has also been implicated in the transition from initiation to replication (10). Preceding initiation of replication, and at a point near the end of mitosis (11), assembly of the MCM proteins into a prereplication complex (pre-RC) occurs dependent on coordinated function of the origin recognition complex (ORC) (12) and proteins Cdc6 (5) and Cdt1 (13). Initiation then depends upon activation by cyclin-dependent kinases (14) and the Dbf4-dependent kinase Cdc7 (15). Evidence derived from temperaturesensitive MCM protein degradation during S-phase (16) and from measurements of MCM protein distribution in chromatin (5) suggests that in S. cerevi...

Identification and Characterization of a Novel Component of the Human Minichromosome Maintenance Complex

“…Similar effects were not observed on MCM6 or MCM7 (data not shown), suggesting that some MCM4 may move on or off chromatin independently of the core helicase complex. While MCM4, -6, and -7 are generally thought to function as a complex, at least two other studies in human cells have found that MCM4 can act independently from MCM6 and MCM7 (16,19). MCM4 has also been shown to assemble on chromatin independently from MCM2 and MCM3 in Xenopus egg extracts (4).…”

“…In human cells, the MCM subunits assemble on DNA beginning in late mitosis and remain associated with the chromatin until early G 2 , after DNA synthesis is completed (11,18,37). MCM proteins have also been shown to be preferentially loaded at human origins of replication and then distributed to more distal sequences during DNA synthesis (16,34). A similar pattern of cell cycle-dependent chromosome and origin association was observed for MCM-BP, consistent with the possibility that some MCM-BP is loaded onto DNA as part of an MCM complex.…”

Androgen Receptor Overexpression in Prostate Cancer Linked to Purα Loss from a Novel Repressor Complex

“…The protein/DNA complexes from intact AD and AI cells or from minced frozen PC tissues were cross-linked and then lysed as previously described (30). Equal amounts of protein or tissue lysates (50-130 mg total weight) were incubated with the primary antibody, Pura 5B11 and 1A12 (31), followed by Protein G PLUS-Agarose beads (Santa Cruz), washed twice with high-salt buffer (0.5 mol/L NaCl), followed by LiCl/detergent solution and TE buffer.…”

Sumoylation of CCAAT/Enhancer-binding Protein α and Its Functional Roles in Hepatocyte Differentiation

“…ChIP Assay and Re-precipitation (Re-ChIP)-Immunoprecipitation of formaldehyde cross-linked chromatin from the transfected SW13, C33a cells, and primary hepatocytes was performed using the ChIP assay kit (Upstate Biotechnology, Inc.) according to the manufacturer's instructions with some modifications (27,28). The immunoprecipitated DNAs and input DNA were analyzed by PCR using the following as primers: 5Ј-CATAACTTTAATGAATTGACAAAGC-3Ј and 5Ј-GAGAAACTCGCTCTAATATACTTCTC-3Ј for the rat albumin promoter; 5Ј-TTTTTGGCAAGGATGGTATG-3Ј and GLprimer2 (Promega) for the promoter region of the luciferase reporter plasmid (albumin promoter); 5Ј-GCACTGTG-CAGAAGAGCTGG-3Ј and 5Ј-CTGCAGCGGCGATCAGA-CCC-3Ј for the rat DHFR promoter; and 5Ј-AGGCTTCCGG-CGAGACATGGC-3Ј and 5Ј-ATGTTCTGGGACACAGCG-ACGATGC-3Ј for the human DHFR promoter.…”