Chromosomes are the permanent repositories of the information that directs eukaryotic cell function. Consistent with this critical role, 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.
Our studies focus 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 assembly of a pair of complex DNA synthesis machines (replisomes) that will go on to replicate the adjacent DNA. The events that occur at origins can be broken down into four steps: origin recognition, helicase loading, helicase activation, and replisome assembly. Helicase loading is tightly restricted to the G1 phase of the cell cycle, whereas helicase activation and replisome assembly occur during S phase. This temporal separation of events provides each origin only one opportunity to initiate replication per cell division cycle.
The six-protein origin recognition complex (ORC) mediates the initial selection of origin DNA sequences. ORC was initially discovered in the budding yeast Saccharomyces cerevisiae, based on its ability to bind to a conserved sequence present in this organism's short (100-120 bp), well characterized origins of DNA replication. This sequence is present in many positions across the genome, yet only a subset of these sites are bound by ORC. In collaboration with David MacAlpine's lab (Duke University), we have used genome-wide mapping of nucleosomes to demonstrate that nucleosome-depleted regions are a critical determinant in the selection of origins of replication. Once bound to origin DNA, ORC is required to establish the precise position of nucleosomes on both sides of the origin. Previous experiments in our lab indicate that this positioning is critical for subsequent helicase loading.
Unlike the situation in S. cerevisiae cells, the sequences that act at origins of replication are not well defined in metazoan cells. This is consistent with the apparent lack of sequence-specific DNA-binding activity for ORC derived from these organisms. To understand how origin selection occurs in metazoan cells, we have used genomic approaches to map ORC-binding sites and replication origins. We initially applied these approaches to the fruit fly Drosophila melanogaster, and found that ORC is localized to specific regions of the genome, many of which act as early origins of replication. We are extending these approaches to human cells, with the goal of combining this information with biochemical studies of metazoan ORC to identify the determinants that mediate ORC localization and origin selection.
As cells enter the G1 phase of the cell cycle, chromatin-associated ORC recruits two other replication factors (Cdc6 and Cdt1) and the eukaryotic DNA helicase (the Mcm2-7 complex) to the origin DNA. In a coordinated series of events, ORC, Cdc6 and Cdt1 act to load multiple DNA helicases onto the adjacent DNA, forming pre-replicative complexes (pre-RCs). Formation of the pre-RC is required to license each potential origin for initiation of replication in the subsequent S phase and is tightly restricted to the G1 phase of the cell cycle (Figure 1).
We have investigated the process of helicase loading in S. cerevisiae cells with a focus on ATP control. Using in vitro assays for these events, we have identified multiple, sequential ATP-dependent steps required for helicase loading that ensure 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 Mcm2-7 onto origin DNA. Thus, Cdc6 can only catalyze helicase loading when it is bound to ORC at the origin. ORC also hydrolyzes ATP, but this event is not required for the initial loading of Mcm2-7. Instead, ORC ATP hydrolysis is required for multiple rounds of helicase loading, ensuring that one round of helicase loading is finished before a second is initiated. These and other observations have led us to propose a model for helicase loading shown in Figure 2.
We are continuing to dissect the events of helicase loading, with a focus on how the ORC/Cdc6/Cdt1 molecular machine alters the affinity of Mcm2-7 helicase for origin DNA. Our studies range from dissecting the function of individual proteins in this process to addressing the regulation of helicase loading during the cell cycle. For example, we have found that the smallest ORC subunit (Orc6) recruits Cdt1 and the Mcm2-7 as a complex to the origin. This is mediated by direct interactions between Orc6 and Cdt1 and, like ORC ATP hydrolysis, this interaction must be dynamic to allow multiple rounds of Mcm2-7 loading. Similarly, we are addressing the mechanisms by which cyclin-dependent kinases regulate the ability of ORC to participate in helicase loading.
All of these events occur in the context of nucleosomal DNA, and we are investigating the role of nucleosomes during helicase loading. Our previous in vivo studies found that local nucleosome position is important for helicase loading, but the mechanistic role for this requirement remains unclear. We have modified the in vitro helicase-loading assay to use nucleosomal templates and are investigating the effects of this change on the reaction. We find that the DNA sequence requirements at the origin are substantially different in the presence of nucleosomes. Currently we are investigating whether these additional DNA requirements influence local nucleosome positioning or are required to recruit factors required for helicase loading in a nucleosomal context. In addition, we are addressing how local nucleosome positioning facilitates helicase loading.
Helicase Activation and Replisome Assembly
Mcm2-7 complexes that are loaded onto the origin DNA during G1 are only activated upon entry into the S phase of the cell cycle. The essential S-phase kinase, Cdc7-Dbf4 (also called Dbf4-dependent kinase, or DDK), triggers a cascade of protein associations required for this activation. We are investigating the mechanisms that control the specificity of DDK action and the consequences of its phosphorylation. Through studies of DDK modification of in vitro assembled pre-RCs, we found that DDK preferentially binds to and phosphorylates a conformationally distinct population of Mcm2-7 complexes that have been loaded onto the origin DNA. Further studies found that this preference requires prior phosphorylation of Mcm4 and Mcm6 by a distinct, unknown kinase that preferentially targets only those complexes that are associated with the origin DNA. These findings provide insight into the mechanisms that target DDK to the origin-bound Mcm2-7 complexes that will drive replication fork movement and suggest new mechanisms to regulate origin activity. Using a combination of approaches, we have identified the sites targeted by both the priming kinase(s) and DDK and are investigating how these modifications modulate Mcm2-7 function and the kinase(s) responsible for the priming phosphorylation.
We are also exploring the events that occur downstream of kinase activation upon entry into S phase. To this end, we are developing assays for the events that occur at the origin after helicase loading, with the goal of developing an in vitro replication initiation assay that uses a defined origin of replication.
These studies are supported in part by the National Institutes of Health.
As of October 27, 2009