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

Abstract

Metazoan genomes contain thousands of replication origins, but only a limited number have been characterized so far. We developed a two-step origin-trapping assay in which human chromatin fragments associated with origin recognition complex (ORC) in vivo were first enriched by chromatin immunoprecipitation. In a second step, these fragments were screened for transient replication competence in a plasmid-based assay utilizing the Epstein-Barr virus latent origin oriP. oriP contains two elements, an origin (dyad symmetry element [DS]) and the family of repeats, that when associated with the viral protein EBNA1 facilitate extrachromosomal stability. Insertion of the ORC-binding human DNA fragments in oriP plasmids in place of DS enabled us to screen functionally for their abilities to restore replication. Using the origin-trapping assay, we isolated and characterized five previously unknown human origins. The assay was validated with nascent strand abundance assays that confirm these origins as active initiation sites in their native chromosomal contexts. Furthermore, ORC and MCM2-7 components localized at these origins during G 1 phase of the cell cycle but were not detected during mitosis. This finding extends the current understanding of origin-ORC dynamics by suggesting that replication origins must be reestablished during the early stages of each cell division cycle and that ORC itself participates in this process.

Phosphorylation of ORC2 Protein Dissociates Origin Recognition Complex from Chromatin and Replication Origins

“…5E). As shown previously with endogenous ORC2 (19), the FLAG-ORC2 wild type protein bound to both origins of replication in G 1 /S phase cells but did not do so in G 2 …”

“…Origin trapping experiments have shown that the human ORC dissociates from the replication origins during the S phase and later in the cell cycle (19). The ORC2-GFP, which is diffusely distributed in nuclear area during early mitosis, binds rapidly to anaphase chromatin in Drosophila embryos (38).…”

“…ORC1 association allows ORC to function at the replication origin, and ORC1 is dissociated from chromatin at the end of the S phase (16 -18). Although the yeast ORC binds to the origin of replication throughout the cell cycle, several reports have suggested that mammalian ORC dissociates from chromatin and the replication origin as the cell progresses through the S phase through some unknown mechanism (15,16,19), but the mechanism of this dissociation is not known.…”

One-way PCR-based mapping of a replication initiation point (RIP)

“…We found that the simplest and most promising approach of isolating nascent DNA strands is: (i) to lyse cells directly in the wells of alkaline agarose gels; (ii) to separate DNA by electrophoresis under denaturing conditions and (iii) to isolate short single-stranded DNA by gel extraction 12 . This procedure minimizes sample handling and has been previously employed by others to map oris in mammalian cells 13,14 . This simple method also appears to minimize DNA damage because origin enrichment was not found with nonreplicating control DNA 10 .…”

Non-coding Y RNAs associate with early replicating euchromatin in concordance with the origin recognition complex

“…Following pre-RC assembly in G1 phase, ORC disassembles partially as ORC1 is degraded while ORC2-6 remains stable and keeps an association with chromatin (Méndez et al, 2002;Tatsumi et al, 2003). The association of human ORC with chromatin and replication origins is regulated during the cell cycle, showing increased association in G1/S compared to G2/M phases (Gerhardt et al, 2006;Prasanth et al, 2004;Siddiqui and Stillman, 2007). ORC3 shows no obvious colocalisation with replication foci suggesting a displacement from replicating chromatin; however, in late S phase cells a weak and apparently euchromatic population of ORC3 remains detectable (see figure 6 of Siddiqui and Stillman, 2007).…”