Flexible DNA Path in the MCM Double Hexamer Loaded on DNA.

The formation of the pre-replicative complex (pre-RC) during the G1 phase, which is also called the licensing of DNA replication, is the initial and essential step of faithful DNA replication during the subsequent S phase. It is widely accepted that in the pre-RC, double-stranded DNA passes through the holes of two ring-shaped minichromosome maintenance (MCM) 2-7 hexamers; however, the spatial organization of the DNA and proteins involved in pre-RC formation is unclear. Here we reconstituted the pre-RC from purified DNA and proteins and visualized the complex using atomic force microscopy (AFM). AFM revealed that the MCM double hexamers formed elliptical particles on DNA. Analysis of the angle of binding of DNA to the MCM double hexamer suggests that the DNA does not completely pass through both holes of the MCM hexamers, possibly because the DNA exited from the gap between Mcm2 and Mcm5. A DNA loop fastened by the MCM double hexamer was detected in pre-RC samples reconstituted from purified proteins as well as those purified from yeast cells, suggesting a higher-order architecture of the loaded MCM hexamers and DNA strands.

Pre-replicative complex assembly with purified proteins.

Licensing of origins of eukaryotic DNA replication involves the loading of six minichromosome maintenance proteins (Mcm2-7) into pre-replicative complexes (pre-RCs). The assembly of the pre-RC is restricted to G1 phase of the cell cycle, which is crucial to ensure once per cell cycle DNA replication. Mcm2-7 is loaded by the action of the origin recognition complex (ORC), Cdc6 and Cdt1 and requires ATP. In vitro reconstitution of this reaction has shown that Mcm2-7 is loaded onto DNA as a symmetrical head-to-head double hexamer. We describe in detail how pre-RC proteins are purified and used to reconstitute pre-RC formation in vitro. This method is useful for studying the biochemical mechanisms of Mcm2-7 loading as well as subsequent steps in DNA replication.

Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila melanogaster.

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.

Multiple functions of the origin recognition complex.

The origin recognition complex (ORC), a heteromeric six-subunit protein, is a central component for eukaryotic DNA replication. The ORC binds to DNA at replication origin sites in an ATP-dependent manner and serves as a scaffold for the assembly of other key initiation factors. Sequence rules for ORC-DNA binding appear to vary widely. In budding yeast the ORC recognizes specific ori elements, however, in higher eukaryotes origin site selection does not appear to depend on the specific DNA sequence. In metazoans, during cell cycle progression, one or more of the ORC subunits can be modified in such a way that ORC activity is inhibited until mitosis is complete and a nuclear membrane is assembled. In addition to its well-documented role in the initiation of DNA replication, the ORC is also involved in other cell functions. Some of these activities directly link cell cycle progression with DNA replication, while other functions seem distinct from replication. The function of ORCs in the establishment of transcriptionally repressed regions is described for many species and may be a conserved feature common for both unicellular eukaryotes and metazoans. ORC subunits were found at centrosomes, at the cell membranes, at the cytokinesis furrows of dividing cells, as well as at the kinetochore. The exact mechanism of these localizations remains to be determined, however, latest results support the idea that ORC proteins participate in multiple aspects of the chromosome inheritance cycle. In this review, we discuss the participation of ORC proteins in various cell functions, in addition to the canonical role of ORC in initiating DNA replication.

A structural role for ATP in the formation and stability of the human origin recognition complex.

The locally restricted recruitment of the multisubunit origin recognition complex (ORC) to eukaryotic chromosomes defines the position of origins of DNA replication. In budding yeast and metazoans the DNA binding activity of ORC is stimulated by ATP and requires an AAA+-type nucleotide binding domain in the largest subunit. Little else is known about the mechanisms behind the ATP requirement for ORC in its initiator function and, specifically, the relevance of nucleotide binding domains present on other subunits. Here we show that ATP is required for specific subunit interactions in the human ORC, with the Orc4 subunit playing a critical role in this dynamic process. ATP is essential for the maintenance of ORC integrity and facilitates complex formation. Thus, besides its previously identified role in DNA binding, ATP serves also as a structural cofactor for human ORC.

Biochemical characterization of two Cdc6/ORC1-like proteins from the crenarchaeon Sulfolobus solfataricus.

The biological role of archaeal proteins, homologous to the eukaryal replication initiation factors of cell division control (Cdc6) and origin recognition complex (ORC1), has not yet been clearly established. The hyperthermophilic crenarchaeon Sulfolobus solfataricus (referred to as Sso) possesses three Cdc6/ORC1-like factors, which are named Sso Cdc6-1, Cdc6-2 and Cdc6-3. This study is a report on the biochemical characterization of Sso Cdc6-1 and Cdc6-3. It has been found that either Sso Cdc6-1 or Cdc6-3 behave as monomers in solutions by gel filtration analyses. Both factors are able to bind to various single-stranded and double-stranded DNA ligands, but Sso Cdc6-3 shows a higher DNA-binding affinity. It has also been observed that either Sso Cdc6-1 or Cdc6-3 inhibit the DNA unwinding activity of the S. solfataricus homo-hexameric mini-chromosome maintenance (MCM)-like DNA helicase (Sso MCM); although they strongly stimulate the interaction of the Sso MCM with bubble-containing synthetic oligonucleotides. The study has also showed, with surface plasmon resonance measurements, that Sso Cdc6-2 physically interacts with either Sso Cdc6-1 or Sso Cdc6-3. These findings may provide important clues needed to understand the biological role that is played by each of these three Cdc6 factors during the DNA replication initiation process in the S. solfataricus cells.