James Berger Faculty Scientist
Assistant Professor of Biochemistry and Molecular Biology
University of California, Berkeley
Department of Molecular and Cell Biology
Research emphasis
Structure/function studies of protein machines and macromolecular complexes
involved in DNA replication, topology, and structure. We study the structural
principles that underlie protein allostery and mediate protein-protein
interactions. Our model systems include type II DNA topoisomerases, helicases,
and replication factors, all of which convert chemical energy into physical
actions that variously manipulate, shuttle, and unravel nucleic acids.
Such reactions have been postulated to require large conformational changes
and complex allosteric mechanisms; however, precise structural definitions
of these events are lacking. Moreover, within the cell these proteins
typically act in the context of large, macromolecular assemblages, a process
that is poorly understood at the molecular level. We study both individual
and multiprotein states of these macromolecules, using biochemical and
structural approaches to derive specific mechanisms for function.
Current projects Type II DNA Topoisomerases. The superhelicity of the DNA is altered
when it is replicated, transcribed, or wrapped about a nucleosome. Likewise,
tangles and knots can arise between circular DNAs or long stretches of
linear DNAs during recombination and replication. Cells use proteins known
as type II DNA topoisomerases to help resolve these topological byproducts.
These proteins by cleaving a single DNA duplex, transporting a second
duplex through the break, and subsequently religating the cleaved DNA.
The reaction mechanism of type II topoisomerases is coupled to ATP binding,
which triggers the cascade of domain movements required for DNA cleavage
and transport. Currently a comprehensive, high-resolution picture of this
reaction cycle is unavailable.
Type II DNA topoisomerases proceed through distinct conformational intermediates
during the course of the duplex transport reaction. We are using X-ray
crystallography to determine the structures of various conformational
states, and of full-length and DNA-bound species, for eukaryotic topoisomerase
II and archaeal topoisomerase VI. Structural information from these studies
will help us to design new biochemical assays for clarifying the general
mechanism of type II topoisomerases, particularly regarding how type II
proteins interact with DNA and how ATP binding drives the conformational
changes required for duplex transport.
Helicases. Helicases are ubiquitous proteins which use the chemical
energy stored in ATP to break base-pairing interactions between nucleic
acid strands and physically separate duplex regions. Helicases are utilized
by the cell in numerous critical processes, including replication, repair,
recombination, splicing, and transcription. The importance of helicases
in these processes is considerable: as mutations in certain helicase families
lead to genetic disorders and cancer, and malfunction in others causes
cell death.
The core mechanism underlying duplex unwinding remains poorly understood.
To explore the mechanisms of helicase function we are using structural
studies of helicases and their regulatory domains that have been trapped
in specific catalytic states by the presence and absence of reaction substrates.
To date, such studies are providing important information regarding regulation
of their helicase catalytic cycle. These data are currently being used
to design and refine additional biochemical experiments with to we will
probe how helicases dynamically couple ATP binding and hydrolysis to allosteric
changes that drive physical motion.
Complex assembly and function. Many of the proteins we study
function in large multiprotein complexes such as the replisome or chromosome
scaffold components. The mechanisms by which these proteins recognize
each other, form specific complexes, and accommodate large internal macromolecular
movements while maintaining stable associations are not understood. We
are characterizing the interactions of helicases and topoisomerases with
other proteins by defining local regions within each enzyme that are critical
for proper complex assembly. This information will be coupled with high-resolution
structural analyses of these regions bound to particular protein components
of the assemblage to dissect the physical basis of complex formation.
Ultimately, structural information from these studies will help determine
how cellular machines such as helicases and type II topoisomerases function
and are regulated within multiprotein contexts as part of their normal
cellular activity.
