First is the assembly of pre-replication complexes (pre-RCs). Knowledge of this process has been greatly aided by the identification of several proteins that bind to replication origins in yeast and the identification of homologues to these proteins in higher eukaryotes. We have shown that the mammalian homologues to these proteins bind to chromatin during telophase and render newly assembled nuclei fully competent to replicate in Xenopus extracts lacking these proteins.

Shortly after the assembly of pre-RCs, a replication timing program is established that determines the order in which chromosomal domains will be replicated (Timing Decision Point; TDP). Intriguingly, we discovered that chromosomal domains become localized to defined positions within the nucleus at the TDP. Our working hypothesis is that the repositioning of sequences at the TDP brings together domains with common functions and common structural proteins. This creates locally high concentrations of specific structural chromatin proteins that in turn influence when replication will initiate.

At a distinct point after the TDP, nuclei experience another transition that specifies the precise sites where replication will initiate (Origin Decision Point; ODP). We are investigating whether the ODP represents a direct molecular change at specific pre-RCs or an epigenetic alteration of chromatin structure that potentiates some pre-RCs while preventing others from initiating. Cells will pass through the ODP whether they are destined to be proliferating (growing and dividing) cells or resting cells. During G1-phase, intra- and extra-cellular signals dictate whether cells will pass through the Restriction point (R-point) and enter S-phase or whether they will arrest and enter a state of quiescence termed Go. If the cell enters Go, specific components of the pre-RC are disassembled, while others appear to remain intact. If conditions are favorable for cell proliferation, a set of cyclin dependent protein kinases are activated to drive cells through the R-point and promote entry into S-phase. We have shown that the ODP takes place prior to and is independent of the R-point. We have also identified inhibitors of specific cellular processes that prevent passage of cells through the ODP, and we have demonstrated that, at one region, transcription is necessary to restrict initiation of replication to specific regions. We are now investigating the hypothesis that pre-RCs are first assembled at many sites during early G1-phase and are eliminated from transcribed regions at the ODP.

The Origin Recognition Complex (ORC) is a hetero-hexameric protein complex that binds to specific replication origin sequences in yeast. All six ORC subunits are highly conserved from yeast to humans, suggesting that the basic mechanism of replication initiation is also conserved. We are studying the structure and function of mammalian ORC in the hopes that it will help us understand how origins are specified. In addition, ORC is required for silencing of specific genes in yeast and appears to be required for the assembly of heterochromatin at certain sites in the chromosomes of flies, suggesting that ORC plays a role in the assembly of silent chromatin that has also been conserved during evolution. We would also like to understand whether and how these two roles of ORC are inter-related.
The figure to the left shows a model resulting from measurements of ORC subunit:chromatin interactions in living cells. We favor a model in which the subunits are rapidly exchanging in and out of the complex throughout the cell-cycle

Upon entry into S-phase, the many enzymes that carry out DNA synthesis are assembled into large macromolecular complexes called replication factories. These factories can be visualized as punctate foci when cells are stained with fluorescent antibodies to replication fork enzymes or nascent DNA. During S-phase, the temporal program for replication proceeds via the coordinated assembly and disassembly of replication factories at different sites within the nucleus. If DNA synthesis is arrested at any point during this process, cellular checkpoints make sure that no further initiation of replication takes place. We have found that these same checkpoints also prevent replication factories from breaking down, so that they will be ready to complete DNA synthesis when conditions return to normal. When the checkpoints are disrupted while DNA synthesis is arrested, cells proceed through the process of assembling new factories and dis-assembling existing factories exactly as in a normal S-phase but in the complete absence of DNA synthesis. We also discovered that the checkpoint stabilizes the DNA primer:template structure itself during the checkpoint arrest period, to allow cells to properly continue replication after the problem is corrected. Many cancer cells have misregulated checkpoint controls, which lead to DNA breaks and further mutations when these replication fork structures collapse. This discovery was later confirmed and dissected genetically in yeast and has turned out to be very important for maintaining genome stability. Interestingly, it is one of the first times that a major conceptual discovery was made first in mammalian cells and later confirmed in yeast - things usually work the other way around.
The figure shows an image of a cell in which replication forks were arrested at sites of early S-phase DNA synthesis (green) yet, because the checkpoint was disrupted, the replication fork protein RPA (red) has moved on to associate with sites that would normally be late replicating at this time. If the checkpoint was not disrupted, the RPA (red) would remain exactly coincident with the arrested replication forks (green) and all sites would be yellow.

All cells contain the same genetic information (DNA) but package it with proteins into "chromatin" in characteristic ways that define each cell type. Chromatin is dismantled and re-assembled during each round of DNA replication, and we have discovered that the temporal order in which segments of DNA are replicated changes as stem cells turn into different cell types. Understanding how to manipulate this packaging process may help us engineer different cell types, a central goal in stem cell therapy.
Replication proceeds via the synchronous firing of clusters of replication origins encompassing domains of several hundred kilobases. These "replication domains" coincide with structural and functional domains of chromosomes and replicate in a defined temporal order during S-phase. Since chromatin is assembled at the replication fork, and since different types of chromatin are assembled at different times during S-phase, it makes sense that replication would be an important regulatory event at which to assemble different types of chromatin in different cell types, but testing this hypothesis has been difficult. Many studies have correlated changes in replication timing to changes in gene expression in different cell lineages and in cancer but none have been able to address the intermediate states that accompany these changes. Mechanistic studies require a system in which these changes can be elicited with sufficient synchrony and homogeneity as to permit biochemical and molecular analyses. We now have such a system at our disposal. We detect dynamic changes in replication timing within a single cell cycle and coincident with key cell fate changes during the differentiation of mouse ES cells to neural precursors. Early to late replication changes coincide with loss of pluripotence and irreversible down-regulation of ES-specific genes, while late to early changes coincide with commitment to neural lineages and up-regulation of neural specific genes. Since replication timing is the only cellular process known to be regulated at the level of large chromosomal domains, our studies have the potential to open a novel chapter in gene regulation. Our working hypothesis is that changes in replication timing during differentiation reinforce the heritability of changes in chromatin structure across large chromosome domains that in turn modulate the responsiveness of genes during stem cell commitment. We are taking both genome-wide "DNA array" approaches to evaluate the significance of replication timing to cell fate changes during differentiation, and more targeted approaches to elucidate the mechanisms relating replication timing and gene expression changes at specific gene loci.