of the Max - MDC

Control of DNA Replication
Manfred Gossen
We are interested in the mechanisms controlling
the initiation of DNA replication in
multicellular organisms. There, the interplay
between chromosomal cis elements and the trans
acting factors (initiators) contributing to replication
initiation is only poorly understood. This
process is the starting point for the ordered execution
of genome duplication and thus crucial for
cellular proliferation and the maintenance of
genome stability. While we initially aim to understanding
basic principles of replication control,
we also start to integrate this research into the
analysis of pathophysiological events, especially
in cancer. The current focus of our work is on the
eukaryotic initiator protein ORC, the origin recognition
complex. Aside from biochemical
approaches, both mammalian tissue cultures and
Drosophila are used as experimental systems. In
addition to the above topics, the group is also
interested in transcriptional cross-talk between
neighboring transgenes as well as the long-term
stability and homogeneity of their expression
patterns.
Localization and cell cycle dynamics of the
Drosophila ORC
Tina Baldinger
ORC is likely to function as the initiator protein in eukaryotes,
i.e. its binding to chromosomal sites specifies the origins
of bi-directional DNA replication. Drosophila
melanogaster offers several distinct advantages for the
analysis of replication initiation factors. Among them are
the availability of a large number of hypomorphic variants
of these proteins and an embryonic development which
relies on maternally supplied stockpiles of replication factors.
To analyze ORC’s binding to chromosomes in vivo, we
generated transgenic flies expressing fully functional GFP
fusions to Orc2, one of the ORC subunits. By genetic complementation
of an Orc2-null background we could determine
the subcellular localization of ORC throughout the cell
cycle, in particular its chromatin association during mitosis
(see Figure). In combination with histone-RFP fusions this
approach revealed changes in the dynamic behavior of ORC
in different tissues and throughout development. It turned
out that the recruitment of ORC to its chromosomal sites is
under the control of the major mediators of cell cycle cues,
the cyclin dependent kinases. These observations will allow
us to directly address the integration of the chromosome
cycle in proliferation control, based on the use of a well
established in vivo model.
Biochemical characterization of the human ORC
Anand Ranjan, Vishal Agrawal
We could co-express the genes for all six subunits of human
ORC in insect cells and purify the resulting protein complex
to homogeneity. Using a Xenopus in vitro replication assay,
the functionality of this recombinant ORC was demonstrated.
It turns out that human ORC is capable of forming various
distinct sub-complexes, which differ in their stability
and DNA binding properties. Interaction studies allowed us
to determine critical features of the architecture of the
human ORC. According to the Saccharomyces cerevisiae paradigm,
ORC’s binding to DNA is expected to be ATP dependent.
We are currently investigating if this unusual mode of
regulating sequence specific DNA interactions is connected
to the ATP dependence of specific subunit interactions that
we observed in our in vitro assays. To this end we are also
testing the biochemical properties of recombinant human
ORC defective in ATP interactions and extend these studies
to human cell cultures. The goal of this project is a better
understanding of the mechanisms by which homologous initiator
proteins like those of yeast and humans accomplish
highly divergent modes of origin determination.
Ablation of preRC proteins
Ibrahim Kocman
ORC binding to chromatin is the first step in the assembly of
the large, origin-associated prereplicative complex, preRC.
This process renders chromosomes replication competent.
All preRC proteins are essential for DNA replication and thus
also for cell proliferation. As such they constitute a potential
target for anti-proliferative therapies, e.g. in the treatment
of cancer. Based on our biochemical analyses we were
able to identify among the preRC genes promising candidates,
which should be particular well suited as a target. In
collaboration with Silence Therapeutics, Berlin, we investi-
Cancer Research 101