A Potential Role for Mini-chromosome Maintenance (MCM) Proteins in Initiation at the Dihydrofolate Reductase Replication Origin

Alexandrow, Mark G.; Ritzi, Marion; Pemov, Alexander; Hamlin, Joyce L.

doi:10.1074/jbc.m108118200

Cited by 26 publications

(20 citation statements)

References 72 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Remnant amounts of Mcm2p-Mcm7p have also been observed by others (Alexandrow et al, 2002;Schaarschmidt et al, 2002). This could be due to the lower concentration of Triton X-100 (0.04%) used to lyse cells, with up to 0.5% being used for others (Kreitz et al, 2001).…”

Section: Hsorc1p Orip-binding Is Cell Cycle Regulatedmentioning

confidence: 87%

Complex protein-DNA dynamics at the latent origin of DNA replication of Epstein-Barr virus

Ritzi

Tillack

Gerhardt

et al. 2003

Journal of Cell Science

Self Cite

100

134

View full text Add to dashboard Cite

The sequential binding of the origin recognition complex (ORC), Cdc6p and the minichromosome maintenance proteins (MCM2-7) mediates replication competence at eukaryotic origins of DNA replication. The latent origin of Epstein-Barr virus, oriP, is a viral origin known to recruit ORC. OriP also binds EBNA1, a virally encoded protein that lacks any activity predicted to be required for replication initiation. Here, we used chromatin immunoprecipitation and chromatin binding to compare the cell-cycle-dependent binding of pre-RC components and EBNA1 to oriP and to global cellular chromatin. Prereplicative-complex components such as the Mcm2p-Mcm7p proteins and HsOrc1p are regulated in a cell-cycle-dependent fashion, whereas other HsOrc subunits and EBNA1 remain constantly bound. In addition, HsOrc1p becomes sensitive to the 26S proteasome after release from DNA during S phase. These results show that the complex protein-DNA dynamics at the viral oriP are synchronized with the cell division cycle. Chromatin-binding and chromatin-immunoprecipitation experiments on G0 arrested cells indicated that the ORC core complex (ORC2-5) and EBNA1 remain bound to chromatin and oriP. HsOrc6p and the MCM2-7 complex are released in resting cells. HsOrc1p is partly liberated from chromatin. Our data suggest that origins remain marked in resting cells by the ORC core complex to ensure a rapid and regulated reentry into the cell cycle. These findings indicate that HsOrc is a dynamic complex and that its DNA binding activity is regulated differently in the various stages of the cell cycle.

show abstract

Section: Hsorc1p Orip-binding Is Cell Cycle Regulatedmentioning

confidence: 87%

Complex protein-DNA dynamics at the latent origin of DNA replication of Epstein-Barr virus

Ritzi

Tillack

Gerhardt

et al. 2003

Journal of Cell Science

Self Cite

100

134

View full text Add to dashboard Cite

show abstract

“…In particular, it is not known whether this complex selectively assembles at initiation zones of DNA replication in mammalian cells. Recent reports have dealt with cell cycle distribution of MCM proteins in chromatin in the hamster DHFR locus (23,24), but these did not specifically address MCM4,6,7 localization or requirements for pre-RC assembly proteins. Another recent paper has examined the cell cycle levels of ORC proteins during the HeLa cell cycle, finding that ORC1 undergoes dynamic changes while the other ORC proteins remain at constant levels (25).…”

Section: Members Of the Minichromosome Maintenance (Mcm)mentioning

confidence: 99%

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

Kinoshita¹,

Johnson

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

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...

show abstract

“…Most well-described origins in vertebrates are characterized by large zones of initiation (28). For example, at the large 55-kb zone of initiation within the dihydrofolate reductase locus, replication bubbles, as well as MCM protein, are detected across the whole intergenic region (3). Origins of this type may better represent the paradigm for cell cycle regulated origins found in metazoans and especially vertebrates.…”

mentioning

confidence: 99%

CpG Methylation of DNA Restricts Prereplication Complex Assembly in Xenopus Egg Extracts

Harvey

Newport

2003

Molecular and Cellular Biology

View full text Add to dashboard Cite

In a Xenopus egg replication system, the origin recognition complex (ORC) does not bind to CpG methylated DNA and DNA replication is inhibited. Insertion of low density CpG DNA of at least 1.2 kb into methylated plasmids rescues both replication and ORC binding. Using this pseudo-origin, we find that ORC binding is restricted to low-CpG-density DNA; however, MCM is loaded onto both weakly and highly methylated DNA and occupies at least ϳ2 kb of DNA. Replication initiates coincident with MCM, and even the most distally bound MCM is associated with sites of replication initiation. These results suggest that in metazoans MCM is loaded onto and initiates replication over a large region distant from ORC.During the G 1 phase of the cell cycle proteins, including ORC the origin recognition complex (cdc6), and the MCMhelicase complex, assemble at multiple sites along DNA, forming prereplication complexes (Pre-RCs) (for a review, see reference 6). During the S phase of the cell cycle, chromatinbound Pre-RCs function as sites for initiation of DNA replication. The assembly of Pre-RCs during G 1 is an ordered process in which ORC recognizes and binds to DNA first and subsequently cdc6 associates with the DNA. The ORC-cdc6 complex then facilitates loading of the MCM-helicase onto DNA. The sequential nature of Pre-RC assembly implies that the initial association of ORC with DNA sites determines where Pre-RCs form along the DNA.In the yeast, Saccharomyces cerevisiae, ORC binds specifically and with high affinity to well-defined DNA consensus sequences distributed along the genome (7, 62). As such, replication in this eukaryote initiates reproducibly from unique DNA sites. The specific association of the S. cerevisiae ORC with origin sites has proven to be extremely useful for defining both the compositional organization of a Pre-RC, as well as dynamic changes in this organization during initiation. For example, because ORC associates specifically with DNA, footprinting techniques can be used to demonstrate structural changes during the G 1 -to S-phase transition (21). Similarly, the "fixed" location of Pre-RCs in S. cerevisiae has allowed replication initiation to be mapped at the nucleotide level (9).Although well-defined origins exist in S. cerevisiae, similar sequence-specific sites have remained elusive in other eukaryotic organisms. Rather, in metazoan somatic cells, sensitive PCR and genetic methods have identified large regions of DNA (2 to 50 kb) where initiation occurs (2, 22). Within these "initiation zones" or origins, initiation occurs at many sites. Similarly, strategies using plasmid maintenance assays in human cells have suggested that any human DNA of adequate length is sufficient for cell cycle-controlled autonomous DNA replication (35). Consistent with these observations is the finding that strict sequence-specific binding of purified Schizosaccharomyces pombe and Drosophila melanogaster ORC has not been detected (15,16).Moreover, in metazoans Pre-RC formation appears to be flexible and under developmental c...

show abstract

A Potential Role for Mini-chromosome Maintenance (MCM) Proteins in Initiation at the Dihydrofolate Reductase Replication Origin

Cited by 26 publications

References 72 publications

Complex protein-DNA dynamics at the latent origin of DNA replication of Epstein-Barr virus

Complex protein-DNA dynamics at the latent origin of DNA replication of Epstein-Barr virus

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

CpG Methylation of DNA Restricts Prereplication Complex Assembly in Xenopus Egg Extracts

Contact Info

Product

Resources

About