Human Mcm proteins at a replication origin during the G1 to S phase transition

Abstract

Previous work with yeast cells and with Xenopus egg extracts had shown that eukaryotic pre-replication complexes assemble on chromatin in a step-wise manner whereby specific loading factors promote the recruitment of essential Mcm proteins at pre-bound origin recognition complexes (ORC with proteins Orc1p-Orc6p). While the order of assembly--Mcm binding follows ORC binding--seems to be conserved in cycling mammalian cells in culture, it has not been determined whether mammalian Mcm proteins associate with ORC-bearing chromatin sites. We have used a chromatin immunoprecipitation approach to investigate the site of Mcm binding in a genomic region that has previously been shown to contain an ORC-binding site and an origin of replication. Using chromatin from HeLa cells in G1 phase, antibodies against Orc2p as well as antibodies against Mcm proteins specifically immunoprecipitate chromatin enriched for a DNA region that includes a replication origin. However, with chromatin from cells in S phase, only Orc2p-specific antibodies immunoprecipitate the origin-containing DNA region while Mcm-specific antibodies immunoprecipitate chromatin with DNA from all parts of the genomic region investigated. Thus, human Mcm proteins first assemble at or adjacent to bound ORC and move to other sites during genome replication.

Identification of New Human Origins of DNA Replication by an Origin-Trapping Assay

“…ChIP experiments with HeLa cells were performed essentially as described previously (60). Briefly, 1 ϫ 10 8 HeLa cells were washed with phosphate-buffered saline (PBS) and treated with 1% formaldehyde in prewarmed medium for 4 min at 37°C.…”

“…2 and 3) suggested that in their native chromosomal context, these replicators are likely licensed during G 1 phase to initiate replication. This prediction was tested by measuring Orc2p enrichment at the potential origins ori6 and ori9 in their native chromosomal contexts in ChIP assays relative to preimmune control precipitates and the input DNA (35,60). In initial experiments with asynchronously proliferating HeLa cells, we found that the origin sequence (UPR) (Fig.…”

The Replicative Regulator Protein Geminin on Chromatin in the HeLa Cell Cycle

“…PCR reactions were performed for 45 cycles with standard settings as recommended by the manufacturer. Sequences and conditions for each primer pair are exactly as those described by Schaarschmidt et al (29). Standard DNA samples were diluted from 30, 3, and 0.3 ng up to 30 pg.…”

“…This origin is located in the region between the two active human genes PRKDC and MCM4. It includes the site where ORC is bound and where the assembly of a prereplication complex is initiated (29,34).…”

Segregation of Replicative DNA Polymerases during S Phase

“…The LB2 gene origin of replication (31) and the origin located in the UPR of the MCM4 gene (32) were utilized in this study, and PCR analysis was performed from the areas shown ( Figs. 1 and 2).…”