Richard and Rhoda Goldman Distinguished Professorship in Biological Sciences Professor of Biochemistry, Biophysics and Structural Biology*
*Affiliate, Division of Genetics, Genomics and Development
We study the mechanisms and regulation of the initiation of DNA replication and gene expression in eukaryotes. We are especially focused on how these processes are coupled to the cell cycle and progression from G1 to S phase in a developmental context. Drosophila chromosomes and papilloma viral DNA provide unique opportunities to learn about chromosomal dynamics. The laboratory uses a combination of biochemistry, structural biology and genetics to address these issues at different levels.
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 animal, plant and fungal cells. ORC helps target the general factors used ubiquitously to unwind and copy DNA. Recent structural work leads us to speculate that ORC must participate in DNA melting much as the prokaryote initiator factor DnaA melts DNA for loading of the processive helicase DnaB. We have purified a complex called CMG that we have proposed as the core machinery for the cellular DNA helicase. How this CMG complex assembles and receives signals from the S phase promoting kinases is now an important issue that we are focusing on.
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 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.
DNA tumor viruses use host cellular proteins for their DNA replication, but encode for site specific DNA binding and helicase activities to sequester the cellular enzymes on their chromosomes and to unwind DNA. We have thus used these viruses to learn about specialized DNA replication initiation. Papillomaviruses oncogenically transform cells, and stably maintain the viral genome in these cells as multicopy nuclear plasmids. The viral encoded DNA binding protein E2 (not to be confused with the cellular factor E2F!), is the master regulatory protein for this class of viruses and has multiple functions for the maintenance of the plasmid in dividing cells. For example E2 brings viral plasmids to mitotic chromosomes to hitch-hike for segregation purposes during cell division. The cellular chromatin binding protein Brd4 interacts with E2 and this binding is necessary for mitotic hitch-hiking. We have recently solved the crystal structure of the E2/Brd4 complex and this structure might help in the development of pan-papilloma drugs useful in curing various precancerous lesions.
E2 has 17 binding sites juxtaposed near five viral promoters in the prototype Bovine virus, BPV-1. The protein stimulates transcription from four of these promoters, and in fact stimulates transcription of its own promoter. The protein also induces transcription of a viral promoter encoding for a trans-repressor. In G0 cells the repressor protein predominates, while in S phase the enhancer protein is induced. The positive factor is a paradigm eukaryote enhancer protein, as its cis sites can stimulate transcription from heterologous promoters in a distance- and orientation-independent manner.
The replication factor (called E1) encoded by these viruses is a 68 kd phosphoprotein. It binds ATP, and assembles to forms a hexameric DNA helicase. Before this assembly E1 associates in a step wise manner to the viral origin site. The E1 monomer protein binds to the origin of DNA replication directly, but requires E2 to help find the cis acting DNA-element. The E2 protein is thus an enhancer of both replication and transcription. Our work shows that E1 can form a complex with the E2 factor and that this protein/protein interaction is critical for cooperative DNA binding to the ori site. The transcription factor thus serves as a molecular chaperone for the initiating helicase. Interestingly the x-ray structure of the E2/E1 complex shows us that the surface critical for that complex is separate from the face of E2 that interacts with Brd4. This work demonstrates that transcription factors can evolve multiple activities in a single domain and we suspect that this may be uncovered for the cellular factors carried within the Myb- complex described in drosophila cells.
Nucleotide-dependent conformational changes in the DnaA-like core of the origin recognition complex (Clarey MG, Erzberger JP, Grob P, Leschziner AE, Berger JM, Nogales E, Botchan M. ). Nat Struct Mol Biol. 2006 Aug; 13(8):684-90.
Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryote DNA replication fork helicase (Moyer SE,Lewis PW, Botchan MR. Proc Natl Acad Sci USA 2006, Jul 5; 103(27):10236-41)
Identification of a Drosophila Myb-E2F2/RBF transcriptional repressor complex ( Lewis PW, Beall EL, Fleischer TC, Georlette D, Link AJ, Botchan MR. Genes Dev. 2004 Dec 1; 18(23):2929-40. 2004)
Dm-myb mutant lethality in Drosophila is dependent upon mip130: positive and negative regulation of DNA replication. ( E.L, Beall, Bell, M, Georlette D., and MR Botchan; Genes Dev. 2004 Jul 15; 18(14):1667-80. )
Role for a Drosophila Myb-containing protein complex in site-specific DNA replication.(Beall EL, Manak JR, Zhou S, Bell M, Lipsick JS, Botchan MR Nature. 2002 Dec 19-26; 420(6917):833-7.
Assembly of functionally active Drosophila origin recognition complex from recombinant proteins. [I. Chesnokov, M. Gossen, D. Remus, and M. Botchan (1999) Genes & Dev. 13, 1289-1296]
DNA topology, not DNA sequence, is a critical determinant for Drosophila ORC-DNA binding(Remus D, Beall EL, Botchan MR EMBO J. 2004 Feb 25; 23(4):897-907. 2004)
The X-ray structure of the papillomavirus helicase in complex with its molecular matchmaker (EAbbate, Berger JM, Botchan MR Genes Dev. 2004; 18(16):1981-96)
Crystal Structure of the Human Papillomavirus Type 18 Activation Domain. [S. Harris and M. Botchan (1999) Science 284,1673-1677]
Biochemical and electron microscopic image analysis of the hexameric E1 helicase. [E. Fouts, X. Yu, E. H. Egelman, and M. R. Botchan (1999) J. Biol. Chem. 274(7), 4447-4458]
Association of the origin recognition complex with heterochromatin and HP1 in higher eukaryotes. [D. T. S. Pak, M. Pflumm, I. Chesnokov, D. W. Huang, R. Kellum, J. Marr, P. Romanowski, and M. Botchan (1997) Cell 91, 311-323]
Last Updated 2006-08-09