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.

Protein microarrays provide a powerful tool for the study of protein function. However, they are not widely used, in part because of the challenges in producing proteins to spot on the arrays. We generated protein microarrays by printing complementary DNAs onto glass slides and then translating target proteins with mammalian reticulocyte lysate. Epitope tags fused to the proteins allowed them to be immobilized in situ. This obviated the need to purify proteins, avoided protein stability problems during storage, and captured sufficient protein for functional studies. We used the technology to map pairwise interactions among 29 human DNA replication initiation proteins, recapitulate the regulation of Cdt1 binding to select replication proteins, and map its geminin-binding domain.

Mcm10 (Dna43), first identified in Saccharomyces cerevisiae, is an essential protein which functions in the initiation of DNA synthesis. Mcm10 is a nuclear protein that is localized to replication origins and mediates the interaction of the Mcm2-7 complex with replication origins. We identified and cloned a human cDNA whose product was structurally homologous to the yeast Mcm10 protein. Human Mcm10 (HsMcm10) is a 98-kDa protein of 874 amino acids which shows 23 and 21% overall similarity to Schizosaccharomyces pombe Cdc23 and S. cerevisiae Mcm10, respectively. The messenger RNA level of HsMcm10 increased at the G(1)/S-boundary when quiescent human NB1-RGB cells were induced to proliferate as is the case of many replication factors. HsMcm10 associated with nuclease-resistant nuclear structures throughout S phase and dissociated from it in G(2) phase. HsMcm10 associated with human Orc2 protein when overexpressed in COS-1 cells. HsMcm10 also interacted with Orc2, Mcm2 and Mcm6 proteins in the yeast two-hybrid system. These results suggest that HsMcm10 may function in DNA replication through the interaction with Orc and Mcm2-7 complexes.

Origin recognition complex (ORC) plays critical roles in the initiation of DNA replication and cell-cycle progression. In metazoans, ORC associates with origin DNA during G1 and with heterochromatin in postreplicated cells. However, what regulates the binding of ORC to chromatin is not understood. We have identified a highly conserved, leucine-rich repeats and WD40 repeat domain-containing protein 1 (LRWD1) or ORC-associated (ORCA) in human cells that interacts with ORC and modulates chromatin association of ORC. ORCA colocalizes with ORC and shows similar cell-cycle dynamics. We demonstrate that ORCA efficiently recruits ORC to chromatin. Depletion of ORCA in human primary cells and embryonic stem cells results in loss of ORC association to chromatin, concomitant reduction of MCM binding, and a subsequent accumulation in G1 phase. Our results suggest ORCA-mediated association of ORC to chromatin is critical to initiate preRC assembly in G1 and chromatin organization in post-G1 cells.

Eukaryotic cells possess overlapping mechanisms to ensure that DNA replication is restricted to the S phase of the cell cycle. The levels of hOrc1p, the largest subunit of the human origin recognition complex, vary during the cell division cycle. In rapidly proliferating cells, hOrc1p is expressed and targeted to chromatin as cells exit mitosis and prereplicative complexes are formed. Later, as cyclin A accumulates and cells enter S phase, hOrc1p is ubiquitinated on chromatin and then degraded. hOrc1p destruction occurs through the proteasome and is signaled in part by the SCF(Skp2) ubiquitin-ligase complex. Other hORC subunits are stable throughout the cell cycle. The regulation of hOrc1p may be an important mechanism in maintaining the ploidy in human cells.

The origin recognition complex (ORC) was initially discovered in budding yeast extracts as a protein complex that binds with high affinity to autonomously replicating sequences in an ATP-dependent manner. We have cloned and expressed the human homologs of the ORC subunits as recombinant proteins. In contrast to other eukaryotic initiators examined thus far, assembly of human ORC in vitro is dependent on ATP binding. Mutations in the ATP-binding sites of Orc4 or Orc5 impair complex assembly, whereas Orc1 ATP binding is not required. Immunofluorescence staining of human cells with anti-Orc3 antibodies demonstrate cell cycle-dependent association with a nuclear structure. Immunoprecipitation experiments show that ORC disassembles as cells progress through S phase. The Orc6 protein binds directly to the Orc3 subunit and interacts as part of ORC in vivo. These data suggest that the assembly and disassembly of ORC in human cells is uniquely regulated and may contribute to restricting DNA replication to once in every cell division cycle.

ORC2 is a subunit of the origin recognition complex in yeast and has been implicated in the initiation of DNA replication and transcriptional silencing. We have isolated mouse and human cDNA clones encoding proteins with 47.9 and 46.3% similarity, respectively, to yeast ORC2. This degree of similarity and the alignment of sequences suggest that these clones may represent a mammalian ORC2 homologue. The existence of such a homologue would, in turn, suggest the existence of a mammalian origin recognition complex, similar to that found in yeast. Although Northern blot analysis of various adult mouse tissues found the highest levels of expression of ORC2-like (ORC2L) RNA in testes, strong signals did not always correspond to tissues in which high levels of DNA replication would be expected. This finding may reflect functional roles of ORC2L distinct from those that it may play in DNA replication. Analyses of somatic cell hybrid DNA and fluorescence in situ hybridization were employed to map the human ORC2L gene to chromosome 2q33.

ORC2 is a subunit of the origin recognition complex in yeast and has been implicated in the initiation of DNA replication and transcriptional silencing. We have isolated mouse and human cDNA clones encoding proteins with 47.9 and 46.3% similarity, respectively, to yeast ORC2. This degree of similarity and the alignment of sequences suggest that these clones may represent a mammalian ORC2 homologue. The existence of such a homologue would, in turn, suggest the existence of a mammalian origin recognition complex, similar to that found in yeast. Although Northern blot analysis of various adult mouse tissues found the highest levels of expression of ORC2-like (ORC2L) RNA in testes, strong signals did not always correspond to tissues in which high levels of DNA replication would be expected. This finding may reflect functional roles of ORC2L distinct from those that it may play in DNA replication. Analyses of somatic cell hybrid DNA and fluorescence in situ hybridization were employed to map the human ORC2L gene to chromosome 2q33.