Eukaryotic DNA replication is a highly conserved process in which orthologs of the proteins responsible for DNA replication in the budding yeast (Saccharomyces cerevisiae) are found in all other eukaryotes 1, 2, 3. At least 35 gene products are required to initiate eukaryotic DNA replication ( Fig. 1 ). Replication begins with the assembly of an origin recognition complex (ORC) composed of six different subunits (Orc1 to Orc6) at DNA replication origins distributed throughout the genome. Thus, it is the interaction between ORC and DNA that determines where DNA replication begins in the genomes of eukaryotic cells. Cdc6 protein binds to ORC/chromatin sites and thereby allows Cdt1(RLF-B) to load Mcm proteins 2 through 7 onto these sites to form a pre-replication complex (pre-RC). These events will not occur in the absence of Noc3, a highly conserved DNA binding protein that is associated with S. cerevisiae replication origins and interacts with ORC and Mcm proteins 4. Mcm2 to Mcm7 exist as a hexameric complex [Mcm(2-7)] that is believed to be the helicase responsible for unwinding parental DNA strands. This process is facilitated by the single-stranded DNA binding trimeric protein complex, RP-A. Presumably, at least two Mcm(2-7) complexes are loaded at each origin of bi-directional replication, although more are possible 5. DNA synthesis (S-phase) is triggered by addition of Mcm10 6 followed by the action of the DNA dependent protein kinase, Cdc7/Dbf4, and a cyclin dependent protein kinase, Cdk2/cyclin E and Cdk2/cyclin A. Both Cdk2 and Cdc7/Dbf4 can phosphorylate Mcm proteins. These events allow Cdc45, Sld, GIN proteins to escort DNA polymerase -a: DNA primase to the pre-RC and initiate RNA-primed DNA synthesis at or close to ORC/chromatin sites.
Figure 1. Proteins and events in the assembly and activation of pre-replication complexes (pre-RCs) on chromatin in the metazoa are indicated above the time line labeled "chromatin". Those events that inactivate pre-RCs are indicated below the time line.
Our current research focuses on the ORC proteins, because of their unique role in determining when (see "ORC Cycle") and where (see "Replication Origins") DNA replication begins. Our goal is to discover communication links between DNA replication and other pathways that regulate cell growth and development. Such links are needed to maintain homeostasis by telling the cell when replication has begun, when it is complete, and when it is aberrant. Previous studies on protein-protein interactions in DNA replication have relied on immuno-precipitation, GST-pull down, and yeast 2-hybrid assays. Our approach is to use constitutively expressed, epitope tagged proteins to purify protein complexes that exist in human cells either during normal cell proliferation or during the transition from G0 to cell proliferation. We hope to identify specific protein-protein interactive domains, mutations and modifications that affect these interactions, and the function of these interactions in DNA replication or cell cycle regulation.
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