(1) The Mechanism of Homologous Recombination
The long-term objective of this research program is to study the mechanism
of human homologous recombination. Currently, we focus on the chromosome
remodeling activity of human Rad54 (hRad54) in the human Rad51 (hRad51) mediated
homologous recombination, and the interactions of p53 with both human Rad51
and human Rad54 that have been postulated to modulate homologous recombination
in response to DNA damage. In addition, we explore possible roles of human
Rad51 in replication fork restart. How does human Rad54 protein remodel
chromosome during recombination? The hRad54 protein serves as an accessory
factor in hRad51 mediated homologous recombination. However, exactly how
hRad54 interacts with hRad51 to stimulate homologous recombination is not
well understood. We are attempting to address the following questions. (I)
Is hRad54 an active component of the nucleoprotein filament formed by hRad51
on ssDNA? (II) If the role of hRad54 in vivo were to induce chromatin remodeling
to facilitate homologous recombination, does the remodeling involve repositioning
(sliding) of nucleosomes or does it involve conformational changes of nucleosomes?
(III) Can hRad54 promote histone octamer transfer? Can hRad54 assist hRad51-ssDNA
to promote strand exchange when the target duplex DNA is part of a nucleosomal
array? We also ask, can hRad51 catalyze replication fork regression?
DNA recombination is considered to be integral to DNA replication and cell
survival. Recombination allows replication to successfully bypass the roadblocks
created by DNA damage or the collapse of a replication fork. Using synthetic
replication forks, we are testing: (I) Can hRad51 catalyze replication fork
regression that has been demonstrated in prokaryotic system? (II) Is there
any role for hRad54 in this process? Finally, we ask does p53 modulate homologous
recombination through its interactions with human Rad51 and human Rad54?
p53 suppresses genome instability, particularly in response to DNA damage.
p53 has been implicated in homologous recombination, and recently has been
shown to interact with hRad51 and hRad54 in vivo. We are testing the hypothesis
that p53 contributes to genome stability by transcription-independent modulation
of homologous recombination, and a stalled replication fork. Specifically,
we try to address: (I) Can p53 inhibit fork regression promoted by hRad51
thus blocking the restart of replication? (II) Can p53 inhibit hRad51 promoted
strand exchange? (III) Does hRad54 play any role in the above processes?
(2) Topological problems of kinetoplast DNA replication
Kinetoplast DNA (kDNA) is the mitochondrial (mt) DNA of trypanosomatids
and related protozoan parasites. The structure of kDNA is unique in nature,
consisting of a network of thousands of topologically interlocked DNA circles,
which resembles the chain mail of medieval armor. There are two types of
DNA circles found in various kDNA networks, typically a few dozen maxicircles
(~ 20-40 kb) and several thousand minicircles about 0.5-3 kb in length. The
complex topology makes the replication of the entire network a unique and
interesting problem in biology. Studies of the last two decades have revealed
that kDNA network replication includes releasing of each minicircle from
the network, replication of the minicircles, segregation of the replicated
daughter minicircles, and finally reattaching them back to the network. After
each of the maxi- and minicircles has been replicated, the network is then
divided into two progeny networks. All these topological transformations
of kDNA network are catalyzed by DNA topoisomerase(s). In theory, although
the topoisomerase can make any topological conversions, there have to be
mechanisms that provide for specificity and for directionality of these conversions.
What factors direct the unlinking and reattachment of minicircles from the
kDNA network during the replication? We attempt to understand the mechanisms
that regulating the directionality of these topological conversions. The
goal of this project is to investigate above question directly by using a
Crithidia fasciculata mitochondrial topoisomerase II (mt Topo II) that we
have recently purified. Specifically, we are trying to address the following
questions: (I). Does the mt Topo II shift the steady state fraction of catenanes
formed in vitro by plasmid DNAs away from thermodynamic equilibrium? What
is the direction of the shift? (II). Does the intrinsically bent segments
of the minicircles affect the topoisomerization reaction catalyzed by mt
Topo II? (III). Do the single-stranded gaps and nicks found in the newly
synthesized minicircles affect the topoisomerization reaction catalyzed by
mt Topo II? (IV). Can mt Topo II catalyze the reconstruction of the two-dimensional
kDNA network with the purified maxicircles and monomer minicircles as the
substrates?
(3) Large-Scale Genomic Monitoring of Ecosystems Using a DNA-based Memory
Collaborating with computer scientist, bioengineer, and environmental
scientist, we are developing and implementing a new methodology for a genome-enabled,
ecological monitoring system.
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