Michael Botchan
Michael BotchanProfessor of Biochemistry and Molecular Biology**And Affiliate, Division of Genetics and Development E-mail: mbotchan@berkeley.edu |
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Full Directory InformationResearch Interests
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We study the mechanisms and regulation of the initiation of DNA replication in eukaryotes and how this process is coupled to the cell cycle. Drosophila chromosomes and papilloma viral DNA provide unique opportunities to learn about chromosomal dynamics.
Current Projects
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The chromosomes of eukaryotic cells contain cis-acting elements important for gene expression, replication, folding and structure, segregation and recombination. Among these regulatory sites only those involved in gene expression are well studied.
In eukaryotes each chromosome has many sites that serve as initiators for DNA replication in dividing cells. For multicellular eukaryotes the utilization of these sites changes during the course of development, and though the program of activation is poorly understood the process is known to affect both gene expression programs and chromosomal folding.
We have purified a complex of six polypeptides from Drosophila embryos that is called the Drosophila origin recognition complex (ORC), as it has structural and biochemical homologies to the well defined budding yeast ORC protein. This complex is part of the machinery that marks chromosomal DNA as sites "to be initiated" for replication. Presently we are developing biochemical and genetic assays to learn in detail how such temporal and spatial regulation for DNA replication initiation is achieved. We have cloned the genes encoding for each of the 6 subunits of the Drosophila ORC and are investigating the DNA binding specificities of the complex. It is well established that ORC persists on chromosomes throughout the cell cycle and in late mitosis/G1 the cdc6 protein associates with ORC creating a preinitiation complex of unknown complexity. We have cloned the Drosophila cdc6 gene and are testing the hypothesis that the activity of cdc6 is regulated by the cdk2:cyclin E kinase.
Most intriguingly we have observed that there is a very high density of ORC-2 protein associated with centric heterochromatin, a region of the chromosome likely to play an important role in chromosome folding, segregation and pairing. It is also known that this compact region of the chromosomes causes severe repression of "euchromatic" genes translocated into this environment, resulting in what is called position effect variegation (PEV). An abundant trans-acting protein known to be important for PEV and heterochromatin folding is called HP-1 and ORC interacts with HP-1. Further it is ORC-1 that has the strongest interaction with HP-1. We have proposed that ORC may have multiple functions in the cell cycle including coordination of folding and unfolding of DNA with the replicationcycle. These ideas are presently being tested through biochemical and genetic means.
DNA tumor viruses use host cell protein for their DNA replication, but encode for site specific DNA binding and helicase activity 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 DNA binding protein E2 brings viral plasmids to mitotic chromosomes to hitch-hike for segregation purposes during cell division.
All papillomaviruses encode for a 45 kd transcription factor called E2. E2 is a site-specific DNA binding protein, and 17 such binding sites are juxtaposed near five viral promoters in the prototype Bovine virus, BPV-1. The protein can stimulate 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 is a DNA helicase. The E1 protein binds to the origin of DNA replication directly, but is aided by E2. 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. The transcription factor thus serves as a molecular chaperone for the initiating helicase. We are presently engaged in structural studies to learn more about the mechanism of action of DNA helicases and how the activation domain of E2 interfaces with both the transcription and replication machinery.
Selected Publications
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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]
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 9/2/2003
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