MERIT Award Recipient: Michael Botchan, Ph.D.
Regulatory Interactions Between Tumor Viruses and Cells
Our laboratory focuses on the regulation and mechanisms of DNA replication. The DNA replication process must be faithful in copying the genetic information in a two-fold way: the machinery that copies the DNA duplex must preserve the integrity of the primary sequence for gene expression, and must also keep the copy number of each gene constant. Any imbalance in copying can lead to dire consequences. Collapse of DNA replication forks, or inappropriate or inefficient assembly of the replication factories at specialized sites called origins of replication, can lead to chromosome breaks and subsequent aneuploidy (incorrect chromosome number) in the cell. These collapses, breaks, and aneuploidies ultimately can lead to cancer.
DNA replication initiation events are characterized by the assembly of special proteins at spots on the DNA called origins of replication, and these proteins in turn lead to the creation of active replication factories. The six protein subunit complex called ORC (for origin recognition complex) is a key factor in this initiation event and the complex is by and large conserved in all animal, plant and fungal cells. ORC helps target the general factors used ubiquitously to unwind and copy DNA. The pattern of origin distribution differs in different cell lineages, but how the "site" choices are established and maintained in any given set of cells and tissues is unknown. ORC has little or no ability to target itself to specific DNA zones, though it is crucial for the actual replication process. Thus, other factors must control this selection process in cell lineage-specific ways. A considerable body of research by many laboratories has shown that regions of the chromosomes containing many genes and/or containing active genes are predictive of the positions of origins of replication, but a mechanistic understanding of the site selection process is still needed.
We have the used fruit fly Drosophila melanogaster to study, in a simple model organism, how such choices are made. In this research we have uncovered a link between proteins that are in the family of human "proto-oncogenes" and tumor suppressors, and the mechanisms by which origin of replication are selected. Specifically, we have shown that the Retinoblastoma (Rb) protein and its DNA docking partners E2F2 and DP, together with the Drosophila Myb protein complex, are important for silencing some potential origins of replication, and allowing others to be used. How do these proteins perform this function? Do they specifically target the ORC to origin regions, or do they work indirectly through modifications of other proteins that in turn allow for the initiation process? Previously, these proteins were known to be involved directly in gene expression patterns, determining which genes are expressed and which genes are not. Given that these factors are known to be important for gene expression, our working hypothesis is that the site-specific DNA binding factors such as Myb and E2Fs act to localize enzymes and remodeling factors that can prepare the chromosome fiber for either DNA replication or gene expression.
Our work has previously focused on understanding ORC localization and assembly of the DNA replication complexes at specific sites in follicle cells that surround the egg. We now wish to ask if these same complexes are involved in the selection of origin of replication sites in other cell types. It was quite unexpected and gratifying to learn that many of the factors we have isolated as a complex in the fruit fly (where they regulate both gene expression and DNA replication) have also been identified, using genetic screens, as important in a possible tumor-suppressor-like function regulating gene expression in nematode worms. Do these proteins also have a dual function and control the selection of replication origins in the worm, as they do in the fruit fly? Do these same complexes have similar functions in human cells? Many of the Myb-associated factors are conserved in human cells, and have been shown to interact with the Rb protein, strengthening that possibility. Future work supported by the NCI should help us understand if origin of replication activity and regulation of gene expression patterns are intimately connected, and if so, the underlying reasons for that connection.