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 replication cycle. 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.
