The origin recognition complex (ORC) is required to initiate eukaryotic DNA replication and also engages in transcriptional silencing in S. cerevisiae. We observed a striking preferential but not exclusive association of Drosophila ORC2 with heterochromatin on interphase and mitotic chromosomes. HP1, a heterochromatin-localized protein required for position effect variegation (PEV), colocalized with DmORC2 at these sites. Consistent with this localization, intact DmORC and HP1 were found in physical complex. The association was shown biochemically to require the chromodomain and shadow domains of HP1. The amino terminus of DmORC1 contained a strong HP1-binding site, mirroring an interaction found independently in Xenopus by a yeast two-hybrid screen. Finally, heterozygous DmORC2 recessive lethal mutations resulted in a suppression of PEV. These results indicate that ORC may play a widespread role in packaging chromosomal domains through interactions with heterochromatin-organizing factors.

The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes. We have reconstituted a functional recombinant Drosophila ORC and compared activities of the wildtype and several mutant ORC variants. Drosophila ORC is an ATPase, and our studies show that the ORC1 subunit is essential for ATP hydrolysis and for ATP-dependent DNA binding. Moreover, DNA binding by ORC reduces its ATP hydrolysis activity. In vitro, ORC binds to chromatin in an ATP-dependent manner, and this process depends on the functional AAA ؉ nucleotide-binding domain of ORC1. Mutations in the ATP-binding domain of ORC1 are unable to support cell-free DNA replication. However, mutations in the putative ATP-binding domain of either the ORC4 or ORC5 subunits do not affect either of these functions. We also provide evidence that the Drosophila ORC6 subunit is directly required for all of these activities and that a large pool of ORC6 is present in the cytoplasm, cytologically proximal to the cell membrane. Studies reported here provide the first functional dissection of a metazoan initiator and highlight the basic conserved and divergent features among Drosophila and budding yeast ORC complexes.Drosophila ORC ͉ DNA replication ͉ ATPase activity ͉ DNA binding T he initiation of DNA replication in higher eukaryotes occurs at thousands of sites along chromosomes, and significant progress has been made in understanding how proteins work to regulate this event such that each region replicates exactly once per cell cycle (1). The origin recognition complex (ORC), a heteromeric six-subunit protein, is a central component for DNA replication. ORC binds to DNA at replication origin sites and serves in some way as a scaffold for assembly of other key initiation factors such as cdc6, cdt1, MCM complex, and cdc45 (2). How these factors eventually lead to a melting of the duplex strands and an engagement of the DNA polymerase alpha is still unknown. A deeper understanding of how the cell cycle machinery triggers initiation and couples this event to signal transduction pathways requires a finer dissection of the initiation process. The initiator role of ORC in the DNA replication process was first discovered in Saccharomyces cerevisiae (3), where special sequences of about 100 base pairs serve as origin (ori) sites (4). Within these elements, a short 11-base pair consensus sequence serves as a core binding site for ORC.Although there is a gratifying conservation of such protein factors in different organisms, a surprising divergence for the cis-acting origin sequences almost certainly underscores evolutionary changes in the way DNA replication is regulated and the addition of roles for replication in sculpting functions of the chromosome. For example, in Saccharomyces pombe, ori sequences appear to be largely asymmetric stretches of AT that do not show consensus sequence elements. DNA binding by ORC may depend on a special N-terminal domain of the ORC4 subunit that contains multiple A͞T hook motifs that can each recognize such DNA ele...

Coordination between separate pathways may be facilitated by the requirements for common protein factors, a finding congruent with the link between proteins regulating DNA replication with other important cellular processes. We report that the smallest of Drosophila origin recognition complex subunits, Orc6, was found in embryos and cell culture localized to the cell membrane and cleavage furrow during cell division as well as in the nucleus. A two-hybrid screen revealed that Orc6 interacts with the Drosophila peanut (pnut), a member of the septin family of proteins important for cell division. This interaction, mediated by a distinct C-terminal domain of Orc6, was substantiated in Drosophila cells by coimmunoprecipitation from extracts and cytological methods. Silencing of Orc6 expression with double-stranded RNA resulted in a formation of multinucleated cells and also reduced DNA replication. Deletion of the C-terminal Orc6 -peanut interaction domain and subsequent overexpression of the Orc6 mutant protein resulted in the formation of multinucleated cells that had replicated DNA. This mutant protein does not localize to the membrane or cleavage furrows. These results suggest that Orc6 has evolved a domain critical mainly for cytokinesis.DNA replication ͉ cytokinesis ͉ peanut

The proteins of the origin recognition complex are found throughout all eukaryotes and have roles beyond that of DNA replication.

Wild-type p53 represses Alu template activity in vitro and in vivo. However, upstream activating sequence elements from both the 7SL RNA gene and an Alu source gene relieve p53-mediated repression. p53 also represses the template activity of the U6 RNA gene both in vitro and in vivo but has no effect on in vitro transcription of genes encoding 5S RNA, 7SL RNA, adenovirus VAI RNA, and tRNA. The N-terminal activation domain of p53, which binds TATA-binding protein (TBP), is sufficient for repressing Alu transcription in vitro, and mutation of positions 22 and 23 in this region impairs p53-mediated repression of an Alu template both in vitro and in vivo. p53's N-terminal domain binds TFIIIB, presumably through its known interaction with TBP, and mutation of positions 22 and 23 interferes with TFIIIB binding. These results extend p53's transcriptional role to RNA polymerase III-directed templates and identify an additional level of Alu transcriptional regulation.

The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of the Orc6 protein which does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.Eukaryotic cells duplicate their genomes with remarkable precision during the course of growth and division. This process depends on stringent regulatory molecular mechanisms that couple DNA replication and cell cycle progression. To efficiently duplicate large genomes, eukaryotes have evolved a mechanism for the initiation of DNA replication that involves multiple origins of replication (ori) along the chromosomal DNA. The utilization of such sites in multicellular organisms changes during development, and this process affects both gene expression and chromosome folding. The program of such spatial and temporal activation is not understood. Although not necessarily random, the origin site selection during early Drosophila and Xenopus development appears to be less dependent on specific DNA sequences (5, 25). In agreement with this idea, a number of studies suggest that specific replicator sequences might be dispensable (22,38,52,53). Later in development origin usage becomes more specific (26, 49) and depends on many mechanisms for selection of the initiation events. Overall, with an exception of the budding yeast Saccharomyces cerevisiae, DNA sequences that define eukaryotic and especially metazoan replication origins are poorly characterized, mainly because of a lack of definitive biochemical or genetic assays (13,17,18).The hexameric origin recognition complex (ORC) is an important component for eukaryotic DNA replication. It was originally discovered in the budding yeast S. cerevisiae, and subsequent studies both in yeast and higher eukaryotes laid the foundation for understanding the functions of this important key initiation factor. ORC binds to origin sites in an ATPdependent manner and serves as a scaffold for the assembly of other initiation factors (3). ORC also directly participates in the loading of initiation factors (6, 45). Sequence rules for ORC DNA binding appear to vary ...

In eukaryotes, DNA replication requires the origin recognition complex (ORC), a six-subunit assembly that promotes replisome formation on chromosomal origins. Despite extant homology between certain subunits, the degree of structural and organizational overlap between budding yeast and metazoan ORC has been unclear. Using 3D electron microscopy, we determined the subunit organization of metazoan ORC, revealing that it adopts a global architecture very similar to the budding yeast complex. Bioinformatic analysis extends this conservation to Orc6, a subunit of somewhat enigmatic function. Unexpectedly, a mutation in the Orc6 C-terminus linked to Meier-Gorlin syndrome, a dwarfism disorder, impedes proper recruitment of Orc6 into ORC; biochemical studies reveal that this region of Orc6 associates with a previously uncharacterized domain of Orc3 and is required for ORC function and MCM2–7 loading in vivo. Together, our results suggest that Meier-Gorlin syndrome mutations in Orc6 impair the formation of ORC hexamers, interfering with appropriate ORC functions.DOI: http://dx.doi.org/10.7554/eLife.00882.001

The origin recognition complex or ORC is a six-subunit protein important for DNA replication and other cell functions. Orc6, the smallest subunit of ORC, is essential for both replication and cytokinesis in Drosophila, and interacts with the septin protein Pnut, which is part of the Drosophila septin complex. In this study, we describe the analysis of the interaction of Orc6 with Pnut and whole Drosophila septin complex. Septin complex was purified from Drosophila embryos and also reconstituted from recombinant proteins. The interaction of Orc6 with the septin complex is dependent on the coiled-coil domain of Pnut. Furthermore, the binding of Orc6 to Pnut increases the intrinsic GTPase activity of the Drosophila septin complex, whereas in the absence of GTP it enhances septin complex filament formation. These results suggest an active role for Orc6 in septin complex function. Orc6 might be a part of a control mechanism directing the cytokinesis machinery during the final steps of mitosis.