The initiation of DNA replication in S phase requires the prior assembly of an origin recognition complex (ORC)-dependent pre-replicative complex on chromatin during G1 phase of the cell division cycle. In human cells, the Orc2 subunit localized to the nucleus as expected, but it also localized to centrosomes throughout the entire cell cycle. Furthermore, Orc2 was tightly bound to heterochromatin and heterochromatin protein 1α (HP1α) and HP1β in G1 and early S phase, but during late S, G2 and M phases tight chromatin association was restricted to centromeres. Depletion of Orc2 by siRNA caused multiple phenotypes. A population of cells showed an S-phase defect with little proliferating cell nuclear antigen (PCNA) on chromatin, although MCM proteins remained. Orc2 depletion also disrupted HP1 localization, but not histone-H3-lysine-9 methylation at prominent heterochromatic foci. Another subset of Orc2-depleted cells containing replicated DNA arrested with abnormally condensed chromosomes, failed chromosome congression and multiple centrosomes. These results implicate Orc2 protein in chromosome duplication, chromosome structure and centrosome copy number control, suggesting that it coordinates all stages of the chromosome inheritance cycle

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Centrosomes, each containing a pair of centrioles, organize microtubules in animal cells, particularly during mitosis. DNA and centrosomes are normally duplicated once prior to 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 Edependent re-duplication of both centrioles and centrosomes in a single cell division cycle. The data suggest that Orc1 is a regulator of centriole and centrosome re-duplication as well as the initiation of DNA replication.

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.Studies on the mechanism of initiation of DNA replication in budding yeast led to the identification of the six-subunit origin recognition complex (ORC) 3 that interacts with and licenses origins of replication prior to S phase (1, 2). The human homologs of the six ORC subunits have been identified and have been shown to interact with each other to form various complexes, suggesting that the licensing function of ORC may be conserved in humans (3-13). Budding yeast ORC binds with moderate affinity and sequence specificity to the autonomously replicating sequences consensus sequence at origins of DNA replication in yeast (14). The identification of specific DNA sequences that define origins of replication in the human genome has not been possible so far, and we sought to generate a biochemical system that would allow us to isolate and test putative DNA sequences that may function as the replicator sequences in human cells. As a first step toward this goal, we set out to establish protocols for the reconstitution of human ORC using a completely recombinant system, using baculovirus expression vectors.The baculovirus expression system has been used successfully to generate recombinant six-subunit assemblies in insect cells, expressing ORC proteins native to Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Drosophila melanogaster (15-17). Work from several groups has shown that human Orc1-Orc5 proteins interact with each other in vivo and can also form a complex in insect cells (6,8,9,11); however, in these experiments, human Orc6 did not associate stoichiometrically. The interactions between human ORC subunits have been mapped, and complex architecture was proposed based on binary interactions between subsets of proteins coexpressed in insect cells (8,10). A recent report showed that ATP plays a role in human ORC assembly, and an Orc4 protein with a mutation in the Walker A motif forms unstable complexes (8). This result was inconsistent with another report that performed a similar...

SummaryWe have investigated the effects of diffusive and convective transport on fibrinolysis. Using a constant pressure drop (ΔP/L) from 0 to 3.7 mmHg/cm-clot to drive fluid permeation, various regimes of lytic agents were delivered into fine and coarse fibrin gels (3 mg/ml) and whole blood clots. Using plasmin (1 μM) delivered into pure fibrin or urokinase (1 μM) delivered into glu-plasminogen (2.2 μM)-laden fibrin, the velocity at which a lysis front moved across fibrin was greatly enhanced by increasing ΔP/L. Lysis of fine and coarse fibrin clots by 1 μM plasmin at ΔP/L of 3.67 and 1.835 mmHg/cm-clot, respectively, led to a 12-fold and 16-fold enhancement of the lysis front velocity compared to lysis without pressure-driven permeation. For uPA-me-diated lysis of coarse fibrin at ΔP/L = 3.67 mmHg/cm-clot, the velocity of the lysis front was 25-fold faster than the lysis front velocity measured in the absence of permeation. Similar permeation-enhanced phenomenon was seen for the lysis of whole blood clots. Without permeation, the placement of a lytic agent adjacent to a clot boundary led to a reaction front that moved at a velocity dependent on the concentration of plasmin or uPA used. Overall, these studies suggest that transport phenomena within the clot can play a major role in determining the time needed for reperfusion during fibrinolysis.

Correction to: The EMBO Journal (2004) 23, 2651–2663. doi:10.1038/sj.emboj.760025