The Origin Recognition Complex Marks a Replication Origin in the Human TOP1 Gene Promoter

Abstract

The locations of the origin recognition complex (ORC) in mammalian genomes have been elusive. We have therefore analyzed the DNA sequences associated with human ORC via in vivo cross-linking and chromatin immunoprecipitation. Antibodies specific for hOrc2 protein precipitate chromatin fragments that also contain other ORC proteins, suggesting that the proteins form multisubunit complexes on chromatin in vivo. A binding region for ORC was identified at the CpG island upstream of the human TOP1 gene. Nascent strand abundance assays show that the ORC binding region coincides with an origin of bidirectional replication. The TOP1 gene includes two well characterized matrix attachment regions. The matrix attachment region elements analyzed contain no ORC and constitute no sites for replication initiation. In initial attempts to use the chromatin immunoprecipitation technique for the identification of additional ORC sites in the human genome, we isolated a sequence close to another actively transcribed gene (TOM1) and an alphoid satellite sequence that underlies centromeric heterochromatin. Nascent strand abundance assays gave no indication that the heterochromatin sequence serves as a replication initiation site, suggesting that an ORC on this site may perform functions other than replication initiation.Origins of DNA replication are the chromosomal regions where DNA replication forks for bidirectional duplication of replicons are established. The large and discontinuous genomes of eukaryotes require a large number of origins that are distributed throughout the genome to guarantee a complete replication within the limited time of the S phase in a cell division cycle (for reviews, see Refs. 1-9. The best characterized eukaryotic origins are those of the budding yeast Saccharomyces cerevisiae because they function as sites of replication initiation in extrachromosomal plasmid DNA and are, therefore, amenable to detailed molecular analyses (10, 11). Prototypic budding yeast origin (autonomously replicating sequences (ARSs) 1 ) are composed of 100 -200 base pairs and contain several essential sequence elements including a domain A with the AT-rich ARS consensus sequence and three short stimulatory elements, B1-B3, which are functionally important but divergent in sequence (12). The ARS consensus sequence and the adjacent B1 domain element constitute a binding site for proteins of the origin recognition complex (ORC), whereas the B3 domain element forms a binding site for the transcription factor Abf1 in some, but not all yeast origins (13).ORC is a multimeric protein complex composed of six essential subunits (Orc1p-Orc6p) that associate in an ATP-dependent manner with ARSs (13-15). The major known function of ORC appears to be the recruitment of factors such as Cdc6, Mcm proteins, and others for the formation of functional prereplication complexes (16 -19).Origins of replication in multicellular organisms can usually not be investigated by ARS assays. Therefore, biochemical procedures such as two-dimensional gel electrop...

Overreplication of short DNA regions during S phase in human cells

“…In agreement with previous observations, we detected a three-to fourfold enrichment of TIMM 5-6 and TOP 5-8 relative to the TIMM 10-11 and TOP 11-12 regions that were used as a baseline (Fig. 1B;Giacca et al 1994;Keller et al 2002). In contrast, when the same nascent strand-enriched fraction was used as a template and cloned DNA was used for normalization, two unexpected results were observed.…”

“…This is consistent with the results for TOP1 and ␤-globin ORIs, where overabundant molecules derive precisely from regions where Orc2 binds and the polarity of nascent strand switches in these two ORIs, respectively (Figs. 2, 5;Aladjem et al 1995;Keller et al 2002). Alternatively, the generation of short molecules could take place somewhere in the vicinity of the replication initiation sites and might not participate in the generation of the mature daughter strands.…”

“…2). We tested whether DNA nascent strands elongated from the overreplicated FUCA1 and GCLC regions by measuring the amount of replication intermediates of different sizes in two gradient fractions and normalizing primer efficiency against total DNA as previously done for the TIMM13 and TOP1 ORIs (Giacca et al 1994;Keller et al 2002). As shown in Supplemental Figure 3B, regions between primers FUC 2-11 and GCLC 19-20 were more abundant than their flanks in a gradient fraction containing replication intermediates up to 800 nt.…”

“…Several of the mammalian ORIs identified to date are close to CpG island promoters (Giacca et al 1994;Delgado et al 1998;Keller et al 2002;Gómez and Brockdorff 2004;Gray et al 2007). However, the well-characterized human ␤-globin ORI (Kitsberg et al 1993) does not colocalize with a CpG island, and examples of ORIs distant from known promoters have also been reported (Mesner and Hamlin 2005;Todorovic et al 2005).…”

Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins

“…In flies, a subset of ORCbinding sites have been identified at loci that promote developmentally regulated DNA amplification through repeated rounds of replication. (23,24) In human cells, certain intergenic regions and CpG islands seem to be enriched in ORC, (25,26) but a consensus binding sequence has not been identified.…”