Overview
We are using a combination of biochemistry, genetics and molecular biology to uncover the mechanisms responsible for the accurate and regulated duplication of eukaryotic chromosomes.

Research Summary
Eukaryotic chromosomes are the permanent repositories of the information that directs cellular events. Consistent with this critical function, chromosome duplication is carefully coordinated with the program of the cell division cycle. Each time a cell divides it must accurately replicate the DNA that forms the foundation of each chromosome and reassemble the proteins that interpret this essential cellular blueprint.

To study this process we have focused on the events that occur at the starting points of chromosome duplication called origins of DNA replication. These DNA sequences are found at multiple sites on each eukaryotic chromosome and direct the formation of a series of multi-protein complexes that mediate chromosome duplication.

DNA replication in S. cerevisiae: The short (100-120 bp), well characterized origins of DNA replication derived from S. cerevisiae chromosomes and the ability to combine biochemical and genetic manipulation have led us to study chromosome duplication in this model eukaryote. During each round of cell division, the cell cycle regulated assembly of multi-protein complexes at the origin DNA culminates in the formation of a bi-directional replication machine called a replisome. These events are nucleated by the recognition of a conserved element within the origin DNA by the six-protein origin recognition complex (ORC). Once bound to the origin, ORC and two other replication factors (Cdc6 and Cdt1) assemble the eukaryotic DNA unwinding enzyme (Mcm2-7 complex) onto the origin DNA. The resulting complex marks all potential sites of replication initiation across the genome and is called the pre-replicative complex (pre-RC). Importantly, the correct regulation of pre-RC formation and activation is essential to ensure that eukaryotic chromosomes are replicated exactly once per cell cycle.

Assembly of DNA Replication Complexes: We are using a combination of genetics, protein biochemistry and in vitro assays for pre-RC formation to determine how this essential complex is assembled and how it prepares the origin DNA for replication initiation. Our recent studies focusing on the ATP control of pre-RC assembly have revealed how multiple ATP-dependent steps required for pre-RC formation are coordinated and ensure that this event occurs at the correct cell cycle time and chromosomal position. ATP hydrolysis by Cdc6 is activated by origin-bound ORC and is required to load the Mcm2-7 onto origin DNA. In contrast, ORC ATP hydrolysis is not required for the initial Mcm2-7 loading event but is required for this event to be repeated. These and other observations have led us to propose a model for pre-RC formation shown in the adjacent figure. Our current efforts are aimed at fully reconstituting pre-RC assembly with purified proteins, testing the predictions of our pre-RC assembly model (e.g., What events trigger ATP hydrolysis by ORC and Cdc6? Does the Mcm2-7 ring encircle dsDNA?), and developing new assays for downstream steps in replication initiation.

Prevention of re-replication: We are investigating the mechanisms that ensure the genome is replicated exactly once per cell cycle. Cyclin-dependent kinase (CDKs) modulation of pre-RC formation and activation is central to this regulation. During S phase, high CDK levels direct pre-RC activation and prevent new pre-RC formation. In contrast, the low CDK levels present during G1 allow pre-RC formation but not activation. ORC is one of three CDK targets whose modification mediates CDK inhibition of pre-RC formation. In collaboration with Fred Cross’s lab (Rockefeller University), we found that the S-phase cyclin Clb5 binds ORC but only after initiation has occurred. Our findings support a model in which Clb5 binding to ORC provides an origin-localized switch that specifically prevents re-initiation at replicated origins.Using mutants in ORC that alter either its Clb5 interaction or its CDK phosphorylation we are determining how these associations/modifications alter ORC’s ability to direct pre-RC formation.

We have also mapped the sites of pre-RC formation and replication initiation in cells that are undergoing re-replication using high-density genomic arrays. We found that only a subset of the origins used in a normal S phase is used during re-replication.  Intriguingly, we also found that not all origins that assemble a pre-RC during re-replication initiate from that site suggesting that the mechanisms that control re-replication target events after pre-RC formation.

Origin Selection and Activation: Replication of the eukaryotic genome is restricted to the S phase of the cell cycle.  Within S phase, each origin of replication initiates replication at a characteristic time. This temporal regulation of initiation is a conserved feature of all eukaryotic genomes, yet we know little about either how or why origin initiation is temporally regulated. We are taking multiple approaches to understand the mechanisms that regulate origin usage in S. cerevisiae. First, we have used both computational and genomic approaches to map origins across the yeast genome at nucleotide resolution. These data have allowed us to perform precise sequence comparisons between different classes of origins (e.g. early and late) revealing previously uncharacterized sequence differences. In addition, we have developed genetic screens to identify genes that influence the time of origin initiation within S phase in the yeast S. cerevisiae. We are now determining how these genes influence the time of initiation with the long-term goal of understanding the cellular importance of maintaining the correct temporal pattern of replication.

Chromosome Duplication in Drosophila melanogaster: We have extended our studies to the fruit fly Drosophila melanogaster to address how origin selection and activation occur in multi-cellular organisms. Unlike the relatively well-defined mechanisms of origin selection in S. cerevisiae, origin selection in multi-cellular organisms is poorly understood. Although ORC still plays a primary role in origin selection, studies of ORC derived from multi-cellular organisms indicate that it has a limited ability to recognize specific DNA sequences. These findings suggest that additional mechanisms influence origin selection in these organisms (e.g. local chromatin structure or chromosomal events).

To investigate the control of origin selection in Drosophila, we have used a genomic microarrays to characterize the replication and transcription dynamics across a chromosome arm. Our studies reveal a defined temporal pattern of replication that correlates with the density of active transcription as measured by RNA Pol II association with the DNA. Our findings indicate that early-replicating domains are defined by the integration of transcriptional status of numerous adjacent genes spanning >100 kb. These data argue that the time of replication initiation is unlikely to be influenced by changes in the expression of individual genes. By combining the results of high resolution mapping of the earliest sites of DNA replication and the identification of ORC binding sites across the chromosome, we have identified 62 new Drosophila origins of replication. Our findings suggest that there are multiple molecular determinants of ORC binding to these origins, including increased AT-content and association with promoter-proximal RNA Pol II binding sites. Our findings have led us to propose that the distribution of transcription across the genome acts locally to influence ORC binding/origin selection and globally to modulate origin activation/replication timing. Our current studies are using the origins we have identified to understand the mechanisms that control the in vivo specificity of ORC DNA association and origin selection. We are also testing the hypothesis that global, but not local, changes in chromatin structure influence replication timing.

Selected Publications
Randell, J. C., Bowers, J. L., Rodriguez, H. K., and Bell, S. P.. Sequential ATP hydrolysis by Cdc6 and ORC directs loading of the Mcm2-7 helicase. Mol Cell 21, 29-39. (2006)

Tanny, R. E., Macalpine, D. M., Blitzblau, H. G., and Bell, S. P. Genome-wide Analysis of Re-replication Reveals Inhibitory Controls That Target Multiple Stages of Replication Initiation. Mol Biol Cell 17, 2415-2423. (2006)

MacAlpine, D. M., Rodriguez, H. K., and Bell, S. P. Coordination of replication and transcription along a Drosophila chromosome. Genes Dev 18, 3094-3105. (2004)

Bowers, J. L., Randell, J. C., Chen, S., and Bell, S. P. ATP Hydrolysis by ORC Catalyzes Reiterative Mcm2-7 Assembly at a Defined Origin of Replication. Mol Cell 16, 967-978. (2004)

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Dr. Bell photo credit: Paul Fetters; image used courtesy of HHMI.

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