ORC Proteins Research Articles

APETALA2/ETHYLENE RESPONSE FACTOR and basic helix–loop–helix tobacco transcription factors cooperatively mediate jasmonate‐elicited nicotine biosynthesis

Transcription factors of the plant-specific apetala2/ethylene response factor (AP2/ERF) family control plant secondary metabolism, often as part of signalling cascades induced by jasmonate (JA) or other elicitors. Here, we functionally characterized the JA-inducible tobacco (Nicotiana tabacum) AP2/ERF factor ORC1, one of the members of the NIC2-locus ERFs that control nicotine biosynthesis and a close homologue of ORCA3, a transcriptional activator of alkaloid biosynthesis in Catharanthus roseus. ORC1 positively regulated the transcription of several structural genes coding for the enzymes involved in nicotine biosynthesis. Accordingly, overexpression of ORC1 was sufficient to stimulate alkaloid biosynthesis in tobacco plants and tree tobacco (Nicotiana glauca) root cultures. In contrast to ORCA3 in C. roseus, which needs only the GCC motif in the promoters of the alkaloid synthesis genes to induce their expression, ORC1 required the presence of both GCC-motif and G-box elements in the promoters of the tobacco nicotine biosynthesis genes for maximum transactivation. Correspondingly, combined application with the JA-inducible Nicotiana basic helix-loop-helix (bHLH) factors that bind the G-box element in these promoters enhanced ORC1 action. Conversely, overaccumulation of JAZ repressor proteins that block bHLH activity reduced ORC1 functionality. Finally, the activity of both ORC1 and bHLH proteins was post-translationally upregulated by a JA-modulated phosphorylation cascade, in which a specific mitogen-activated protein kinase kinase, JA-factor stimulating MAPKK1 (JAM1), was identified. This study highlights the complexity of the molecular machinery involved in the regulation of tobacco alkaloid biosynthesis and provides mechanistic insights about its transcriptional regulators.

Molecular determinants of origin discrimination by Orc1 initiators in archaea

Unlike bacteria, many eukaryotes initiate DNA replication from genomic sites that lack apparent sequence conservation. These loci are identified and bound by the origin recognition complex (ORC), and subsequently activated by a cascade of events that includes recruitment of an additional factor, Cdc6. Archaeal organisms generally possess one or more Orc1/Cdc6 homologs, belonging to the Initiator clade of ATPases associated with various cellular activities (AAA+) superfamily; however, these proteins recognize specific sequences within replication origins. Atomic resolution studies have shown that archaeal Orc1 proteins contact double-stranded DNA through an N-terminal AAA+ domain and a C-terminal winged-helix domain (WHD), but use remarkably few base-specific contacts. To investigate the biochemical effects of these associations, we mutated the DNA-interacting elements of the Orc1-1 and Orc1-3 paralogs from the archaeon Sulfolobus solfataricus, and tested their effect on origin binding and deformation. We find that the AAA+ domain has an unpredicted role in controlling the sequence selectivity of DNA binding, despite an absence of base-specific contacts to this region. Our results show that both the WHD and ATPase region influence origin recognition by Orc1/Cdc6, and suggest that not only DNA sequence, but also local DNA structure help define archaeal initiator binding sites.

Pre-replication complex proteins assemble at regions of low nucleosome occupancy within the Chinese hamster dihydrofolate reductase initiation zone

Genome-scale mapping of pre-replication complex proteins has not been reported in mammalian cells. Poor enrichment of these proteins at specific sites may be due to dispersed binding, poor epitope availability or cell cycle stage-specific binding. Here, we have mapped sites of biotin-tagged ORC and MCM protein binding in G1-synchronized populations of Chinese hamster cells harboring amplified copies of the dihydrofolate reductase (DHFR) locus, using avidin-affinity purification of biotinylated chromatin followed by high-density microarray analysis across the DHFR locus. We have identified several sites of significant enrichment for both complexes distributed throughout the previously identified initiation zone. Analysis of the frequency of initiations across stretched DNA fibers from the DHFR locus confirmed a broad zone of de-localized initiation activity surrounding the sites of ORC and MCM enrichment. Mapping positions of mononucleosomal DNA empirically and computing nucleosome-positioning information in silico revealed that ORC and MCM map to regions of low measured and predicted nucleosome occupancy. Our results demonstrate that specific sites of ORC and MCM enrichment can be detected within a mammalian intitiation zone, and suggest that initiation zones may be regions of generally low nucleosome occupancy where flexible nucleosome positioning permits flexible pre-RC assembly sites.

Identification of novel factors involved in or regulating initiation of DNA replication by a genome-wide phenotypic screen in Saccharomyces cerevisiae

DNA replication in eukaryotic cells is tightly regulated to ensure faithful inheritance of the genetic material. While the replicators, replication origins and many replication-initiation proteins in Saccharomyces cerevisiae have been identified and extensively studied, the detailed mechanism that controls the initiation of DNA replication is still not well understood. It is likely that some factors involved in or regulating the initiation of DNA replication have not been discovered. To identify novel DNA replication-initiation proteins and their regulators, we developed a sensitive and comprehensive phenotypic screen by combining several established genetic strategies including plasmid loss assays with plasmids containing a single versus multiple replication origins and colony color sectoring assays. We isolated dozen of mutants in previously known initiation proteins and identified several novel factors, including Ctf1p Ctf3p, Ctf4p, Ctf18p, Adk1p and Cdc60p, whose mutants lose plasmid containing a single replication origin at high rates but lose plasmid carrying multiple replication origins at lower rates. We also show that overexpression of replication initiation proteins causes synthetic dosage lethality or growth defects in ctf1 and ctf18 mutants and that Ctf1p and Ctf18p physically interact with ORC, Cdt1p and MCM proteins. Furthermore, depletion of both Ctf1p and Ctf18p prevents S phase entry, retards S phase progression, and reduces pre-RC formation during the M-to-G1 transition. These data suggest that Ctf1p and Ctf18p together play important roles in regulating the initiation of DNA replication.

Study of DNA replication in Drosophila using cell free in vitro system

Using Drosophila early egg extracts we have developed an optimized cell free system to study DNA replication. The efficiency of replication depends on a cold treatment of Drosophila embryos before the extract preparation and a formation of nuclei facilitated by the addition of membrane fractions to the extracts. In vitro DNA replication is ORC and CDC6 dependent, as a removal of these proteins from the extracts abolishes DNA replication. The N-terminal part of Orc1 protein, which is important for non-replicative functions of ORC, is dispensable for the replication in vitro. We also show that the conserved ATPase motif of CDC6 is crucial for the replication. Our studies indicate that a Drosophila cell free system proves to be an extremely useful tool for a functional dissection of the processes and factors involved in DNA replication in metazoans.

14-3-3 proteins function in the initiation and elongation steps of DNA replication inSaccharomyces cerevisiae

14-3-3s are highly conserved abundant eukaryotic proteins essential for viability, at least in lower eukaryotes. We previously showed that they associate with mammalian and yeast replication origins in a cell-cycle-dependent manner, and are involved in the initiation of DNA replication. Here, we present evidence that 14-3-3 proteins are novel regulators of the initiation and elongation steps of DNA replication in Saccharomyces cerevisiae. The results show that the Bmh2 protein, one of the two 14-3-3 homologues in S. cerevisiae, interacts with Mcm2 and Orc2 proteins, binds to ARS1 maximally at the G1 phase, is essential for plasmid stability, and is required for normal S-phase entry and progression. Furthermore, during G1 phase, the Bmh2 protein is required for the association of MCM proteins with chromatin and their maintenance at replication origins. The results reveal that 14-3-3 proteins function as essential factors for the assembly and maintenance of the pre-replication complex during G1 phase.

Trypanosome Prereplication Machinery Contains a Single Functional Orc1/Cdc6 Protein, Which Is Typical of Archaea

In unicellular eukaryotes, such as Saccharomyces cerevisiae, and in multicellular organisms, the replication origin is recognized by the heterohexamer origin recognition complex (ORC) containing six proteins, Orc1 to Orc6, while in members of the domain Archaea, the replication origin is recognized by just one protein, Orc1/Cdc6; the sequence of Orc1/Cdc6 is highly related to those of Orc1 and Cdc6. Similar to Archaea, trypanosomatid genomes contain only one gene encoding a protein named Orc1. Since trypanosome Orc1 is also homologous to Cdc6, in this study we named the Orc1 protein from trypanosomes Orc1/Cdc6. Here we show that the recombinant Orc1/Cdc6 from Trypanosoma cruzi (TcOrc1/Cdc6) and from Trypanosoma brucei (TbOrc1/Cdc6) present ATPase activity, typical of prereplication machinery components. Also, TcOrc1/Cdc6 and TbOrc1/Cdc6 replaced yeast Cdc6 but not Orc1 in a phenotypic complementation assay. The induction of Orc1/Cdc6 silencing by RNA interference in T. brucei resulted in enucleated cells, strongly suggesting the involvement of Orc1/Cdc6 in DNA replication. Orc1/Cdc6 is expressed during the entire cell cycle in the nuclei of trypanosomes, remaining associated with chromatin in all stages of the cell cycle. These results allowed us to conclude that Orc1/Cdc6 is indeed a member of the trypanosome prereplication machinery and point out that trypanosomes carry a prereplication machinery that is less complex than other eukaryotes and closer to archaea.

DNA replication and the GINS complex: localization on extended chromatin fibers

BackgroundThe GINS complex is thought to be essential for the processes of initiation and elongation of DNA replication. This complex contains four subunits, one of which (Psf1) is proposed to bind to both chromatin and DNA replication-associated proteins. To date there have been no microscopic analyses to evaluate the chromatin distribution of this complex. Here, we show the organization of GINS complexes on extended chromatin fibers in relation to sites of DNA replication and replication-associated proteins.ResultsUsing immunofluorescence microscopy we were able to visualize ORC1, ORC2, PCNA, and GINS complex proteins Psf1 and Psf2 bound to extended chromatin fibers. We were also able to detect these proteins concurrently with the visualization of tracks of recently replicated DNA where EdU, a thymidine analog, was incorporated. This allowed us to assess the chromatin association of proteins of interest in relation to the process of DNA replication. ORC and GINS proteins were found on chromatin fibers before replication could be detected. These proteins were also associated with newly replicated DNA in bead-like structures. Additionally, GINS proteins co-localized with PCNA at sites of active replication.ConclusionIn agreement with its proposed role in the initiation of DNA replication, GINS proteins associated with chromatin near sites of ORC binding that were devoid of EdU (absence of DNA replication). The association of GINS proteins with PCNA was consistent with a role in the process of elongation. Additionally, the large size of our chromatin fibers (up to approximately 7 Mb) allowed for a more expansive analysis of the distance between active replicons than previously reported.

Initiation at the human Fragile X (FMR1) origin of chromosomal replication

Eukaryotic chromosomal DNA replication begins at origins marked by pre‐replication complexes (pre‐RCs) containing ORC, MCM2‐7, and other proteins. However, understanding of mammalian origins and their possible role in maintenance of genomic stability remains incomplete. We have mapped a replication origin 5' of the FMR1 gene, a heritable fragile site on the human X chromosome. As expected, the FMR1 origin binds to pre‐RC proteins in vivo. FMR1 alleles with heritable CGG repeat expansions suffer chromosome breaks and gaps when cells experience folate deficiency, but how the expansion leads to chromosome fragility is not clear.Recent clues from the human DNA helicase HDHB may lead to one answer for this question. In late M/G1, HDHB assembles in pre‐RCs at the laminB2, MCM4, and FMR1 origins, but not in flanking sequences. Based on siRNA and dominant negative approaches, HDHB is important for initiation. At G1/S, HDHB is released from the laminB2 and MCM4 origins and much of it is exported from the nucleus. However, a low level of HDHB is retained on bulk chromatin during S phase and this fraction is increased in cells exposed to agents that stall fork progression. To ask whether HDHB may localize at stalled replication forks even in unperturbed cells, we used ChIP to study the FMR1 origin region and the common fragile site FRA16D. HDHB was strongly enriched in S phase at both sites and not in flanking regions. Taken together, the data suggest that HDHB functions in initiation of replication at origins and in preventing or repairing stalled replication forks that arise in specific regions even in unperturbed cells.

Identification of novel mutations in theSLC25A15gene in hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome: A clinical, molecular, and functional study

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is an autosomal recessive disorder of the urea cycle. With the exception of the French-Canadian founder effect, no common mutation has been detected in other populations. In this study, we collected 16 additional HHH cases and expanded the spectrum of SLC25A15/ORC1 mutations. Eleven novel mutations were identified including six new missense and one microrearrangement. We also measured the transport properties of the recombinant purified proteins in reconstituted liposomes for four new and two previously reported missense mutations and proved that the transport activities of these mutant forms of ORC1 were reduced as compared with the wild-type protein; residual activity ranged between 4% and 19%. Furthermore, we designed three-dimensional (3D)-modeling of mutant ORC1 proteins. While modeling the changes in silico allowed us to obtain new information on the pathomechanisms underlying HHH syndrome, we found no clear-cut genotype-phenotype correlations. Although patient metabolic alterations responded well to low-protein therapy, predictions concerning the long-term evolution of HHH syndrome remain uncertain. The preference for a hepatic rather than a neurological presentation at onset also continues, largely, to elude us. Neither modifications in oxidative metabolism-related energy, such as those expected in different mtDNA haplogroups, nor sequence variants in SLC25A2/ORC2 seem to be crucial. Other factors, including protein stability and function, and ORC1-ORC2 structural interactions should be further investigated.

Arabidopsis ORC1 is a PHD-containing H3K4me3 effector that regulates transcription

Control of gene expression depends on a complex and delicate balance of various posttranslational modifications of histones. However, the relevance of specific combinations of histone modifications is not fully defined. Downstream effector proteins recognize particular histone modifications and transduce this information into gene expression patterns. Methylation of histone H3 at lysine 4 (H3K4me) is a landmark of gene expression control in eukaryotes. Its recognition depends on the presence in the effector protein of a motif termed plant homeodomain (PHD) that specifically binds to H3K4me3. Here, we establish that Arabidopsis ORC1, the large subunit of the origin recognition complex involved in defining origins of DNA replication, functions as a transcriptional activator of a subset of genes, the promoters of which are preferentially bound by ORC1. Arabidopsis ORC1 contains a PHD and binds to H3K4me3. In addition to H4 acetylation, ORC1 binding correlates with increased H4K20me3 in the proximal promoter region of ORC1 targets. This suggests that H4K20me3, unlike in animal cells, is associated with transcriptional activation in Arabidopsis. Thus, our data provide a molecular basis for the opposite role of ORC1 in transcriptional activation in plants and repression in animals. Since only ORC1 proteins of plant species contain a PHD, we propose that plant ORC1 constitutes a novel class of H3K4me3 effector proteins characteristic of the plant kingdom.

Orc1 Controls Centriole and Centrosome Copy Number in Human Cells

Centrosomes, each containing a pair of centrioles, organize microtubules in animal cells, particularly during mitosis. DNA and centrosomes are normally duplicated once before cell division to maintain optimal genome integrity. We report a new role for the Orc1 protein, a subunit of the origin recognition complex (ORC) that is a key component of the DNA replication licensing machinery, in controlling centriole and centrosome copy number in human cells, independent of its role in DNA replication. Cyclin A promotes Orc1 localization to centrosomes where Orc1 prevents Cyclin E-dependent reduplication of both centrioles and centrosomes in a single cell division cycle. The data suggest that Orc1 is a regulator of centriole and centrosome reduplication as well as the initiation of DNA replication.

Proteins of the origin recognition complex (ORC) and DNA topoisomerases on mammalian chromatin

BackgroundThe process of DNA replication requires the separation of complementary DNA strands. In this process, the unwinding of circularly closed or long DNA duplices leads to torsional tensions which must be released by topoisomerases. So topoisomerases play an important role in DNA replication. In order to provide more information about topoisomerases in the initiation of mammalian replication, we investigated whether topoisomerases occur close to ORC in the chromatin of cultured human HeLa cells.ResultsWe have used different cell fractionation procedures, namely salt and nuclease treatment of isolated nuclei as well as formaldehyde-mediated cross-linking of chromatin, to investigate the distribution of topoisomerases and proteins of the origin recognition complex (ORC) in the chromatin of human HeLa cells. First we obtained no evidence for a physical interaction of either topoisomerase I or topoisomerase II with ORC. Then we found, however, that (Orc1-5) and topo II occurred together on chromatin fragments of 600 and more bp lengths. At last we showed that both topo II and Orc2 protein are enriched near the origin at the human MCM4 gene, and at least some of the topo II at the origin is active in proliferating HeLa cells. So taken together, topoisomerase II, but not topoisomerase I, is located close to ORC on chromatin.ConclusionTopoisomerase II is more highly expressed than ORC proteins in mammalian cells, so only a small fraction of total chromatin-bound topoisomerase II was found in the vicinity of ORC. The precise position of topo II relative to ORC may differ among origins.

Differential association of Orc1 and Sir2 proteins to telomeric domains in Plasmodium falciparum

Telomeres have the capacity to recruit proteins that facilitate the spreading of heterochromatin into subtelomeric DNA regions. In the human protozoan pathogen Plasmodium falciparum, the telomere-associated protein Sir2 has been shown to control the silencing of members of virulence genes at some, but not all, chromosome-end loci, indicating that additional proteins are involved in telomere position effect. Here, we identified, in P. falciparum, a novel telomere-associated protein that displays homology with the origin-of-recognition-complex 1 protein Orc1. Antibodies raised against this P. falciparum protein localized to telomeric clusters in the nuclear periphery and the nucleolus. It was found that, prior to DNA replication, P. falciparum Orc1 and Sir2 undergo drastic subcellular reorganization, such as dissociation from the telomere cluster and spreading into the nucleus and parasite cytoplasm. Relocation of Orc1 and Sir2 was also linked to the partial dissociation of telomere clusters. Super gel-shift and chromatin-immunoprecipitation experiments showed the physical association of Orc1 with telomere repeats but revealed a differential association with adjacent non-coding repeat DNA elements. Our data suggest that Plasmodium telomeres might fold back and that Orc1 cooperates with Sir2 in telomeric silencing.

RNA interference targeting ORC1 gene suppresses the proliferation of vascular smooth muscle cells in rats

Background The proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the pathogenesis of vascular diseases such as atherosclerosis and postangioplasty restenosis. The largest subunit of the origin recognition complex (ORC), ORC1, plays a critical role during the initiation of DNA replication in eukaryotes. However, the involvement of ORC1 in the initiation of DNA replication in VSMCs has not been studied yet. Objective The aim of this study was to silence ORC1 gene selectively by using RNA interference and analyze the effects of ORC1 gene on the proliferation and apoptosis of rat VSMCs. Methods Freshly isolated rat VSMCs were transfected with siRNA targeting ORC1 gene capsulated in liposome. ORC1 protein expression was determined by Western blotting and ORC1 mRNA level by RT-PCR. DNA synthesis was analyzed by 3H thymidine ( 3H-TdR) incorporation and cell proliferative activity and cell cycle distribution by flow cytometry. Two apoptosis-related proteins, Bax and Bcl-2, were examined immunohistochemically. Results Down-regulation of ORC1 mRNA and protein expression was observed in rat VSMCs at 24 h after transfection with the three pairs of siRNA targeting ORC1 gene and this reduction persisted at least 7 days post-transfection. Down-regulation of ORC1 mRNA (60%) and protein (80%) expression was observed at 72 h post-transfection in the cells transfected with B-ORC1 siRNA. A significant decrease in 3H thymidine incorporation was observed in rat VSMCs with ORC1 gene silencing after serum challenge, but not in the non-silenced control. A significant increase in the proliferation index and a significant decrease in the percentage of cells at G 0/G 1 phase after serum challenge were observed in the non-silenced control, but not in ORC1 gene silenced cells. A significant increase in the ratio of Bcl-2/Bax was observed after serum challenge in the non-silenced control, but only a slight increase was found in the ORC1 gene silenced cells. ORC1 gene silencing disappeared 7 days after transfection. Continuous serum challenge stimulated VSMCs to synchronously reenter the cell cycle as evidenced by increases in [ 3H] thymidine incorporation, the proliferation index, and the ratio of Bcl-2/Bax, as non-silenced cells were induced to resume cell cycle progression by the addition of 15% fetal bovine serum to the culture medium. Conclusion ORC1 gene silencing causes rat VSMCs to enter a reversible G 0 quiescent, growth arrested state; thus, ORC1 gene may be an important new target for suppressing VSMCs proliferation.

Butyrate Induced Cell Cycle Arrest in Bovine Cells through Targeting Gene Expression Relevant to DNA Replication Apparatus

Using real-time RT-PCR and Western blot analysis in bovine kidney epithelial cells, we systematically investigated the effects of butyrate on patterns of gene expression relevant to DNA replication apparatus. The real-time PCR and Western blot data generally confirmed previously reported microarray data. Of the five genes tested by quantitative RT-PCR, CDKN1A (p21(waf1)) was up regulated, CDC2/cdk1, MCM6, ORC1L were down regulated, while ORC3L expression remained unchanged following butyrate treatment. Also consistent with RT-PCR results, Western blot analysis confirmed that butyrate up-regulated cyclin-kinase inhibitor p21(waf1) in a does-dependent manner. In contrast, butyrate treatment had no effect on the expression of ERK 1/2 proteins. Also consistent with mRNA results, ORC1 and MCM3 proteins were down-regulated by butyrate treatment, while ORC2 protein remained unchanged. The present results suggest that ORC1, not ORC2 or ORC3, along with MCM proteins play a critical role in regulating the initiation of DNA replication and cell cycle progression in MDBK cells and are targets of butyrate regulation.

Conservation of Epigenetic Regulation, ORC Binding and Developmental Timing of DNA Replication Origins in the Genus Drosophila

There is much interest in how DNA replication origins are regulated so that the genome is completely duplicated each cell division cycle and in how the division of cells is spatially and temporally integrated with development. In the Drosophila melanogaster ovary, the cell cycle of somatic follicle cells is modified at precise times in oogenesis. Follicle cells first proliferate via a canonical mitotic division cycle and then enter an endocycle, resulting in their polyploidization. They subsequently enter a specialized amplification phase during which only a few, select origins repeatedly initiate DNA replication, resulting in gene copy number increases at several loci important for eggshell synthesis. Here we investigate the importance of these modified cell cycles for oogenesis by determining whether they have been conserved in evolution. We find that their developmental timing has been strictly conserved among Drosophila species that have been separate for approximately 40 million years of evolution and provide evidence that additional gene loci may be amplified in some species. Further, we find that the acetylation of nucleosomes and Orc2 protein binding at active amplification origins is conserved. Conservation of DNA subsequences within amplification origins from the 12 recently sequenced Drosophila species genomes implicates members of a Myb protein complex in recruiting acetylases to the origin. Our findings suggest that conserved developmental mechanisms integrate egg chamber morphogenesis with cell cycle modifications and the epigenetic regulation of origins.

Structural Basis of DNA Replication Origin Recognition by an ORC Protein

DNA replication in archaea and in eukaryotes share many similarities. We report the structure of an archaeal origin recognition complex protein, ORC1, bound to an origin recognition box, a DNA sequence that is found in multiple copies at replication origins. DNA binding is mediated principally by a C-terminal winged helix domain that inserts deeply into the major and minor grooves, widening them both. However, additional DNA contacts are made with the N-terminal AAA+ domain, which inserts into the minor groove at a characteristic G-rich sequence, inducing a 35 degrees bend in the duplex and providing directionality to the binding site. Both contact regions also induce substantial unwinding of the DNA. The structure provides insight into the initial step in assembly of a replication origin and recruitment of minichromosome maintenance (MCM) helicase to that origin.

Mouse pre-replicative complex proteins colocalise and interact with the centrosome

Initiation of eukaryotic DNA replication is achieved by the sequential binding of different proteins to origins of DNA replication. Using EGFP-tagged initiator proteins and immunofluorescence techniques we found that most of the ORC and the MCM subunits are localised at centrosomes and are colocalised with the polo-like protein kinase, Plk1. Yeast two-hybrid studies revealed interactions of Plk1 with the Mcm2 as well as the Orc2 protein. Co-immunoprecipitations showed an interaction of Plk1 with Mcm2 as well as interactions of γ-tubulin with Mcm3 and Orc2, respectively. An in vitro phosphorylation assay showed that the Orc2 protein is a substrate of Plk1. Depletion of Orc2 and Mcm3 by siRNA leads to an inhibition of cell proliferation, an altered cell cycle distribution as well as to multinucleated cells with insufficiently organised microtubules. These results indicate an important role of the MCM and ORC proteins in mitosis besides their described role in the establishment of the pre-replicative complex.

The BAH domain facilitates the ability of human Orc1 protein to activate replication origins in vivo

Selection of initiation sites for DNA replication in eukaryotes is determined by the interaction between the origin recognition complex (ORC) and genomic DNA. In mammalian cells, this interaction appears to be regulated by Orc1, the only ORC subunit that contains a bromo-adjacent homology (BAH) domain. Since BAH domains mediate protein-protein interactions, the human Orc1 BAH domain was mutated, and the mutant proteins expressed in human cells to determine their affects on ORC function. The BAH domain was not required for nuclear localization of Orc1, association of Orc1 with other ORC subunits, or selective degradation of Orc1 during S-phase. It did, however, facilitate reassociation of Orc1 with chromosomes during the M to G1-phase transition, and it was required for binding Orc1 to the Epstein-Barr virus oriP and stimulating oriP-dependent plasmid DNA replication. Moreover, the BAH domain affected Orc1's ability to promote binding of Orc2 to chromatin as cells exit mitosis. Thus, the BAH domain in human Orc1 facilitates its ability to activate replication origins in vivo by promoting association of ORC with chromatin.