Our interests are in the molecular mechanisms of
DNA replication, recombination and repair, using bacteriophage
T4 and Escherichia coli as model systems. Many
of our studies involve the fate of blocked or broken
replication forks, where these three processes are
tightly interconnected and interdependent.
In one project, we are investigating the molecular
mechanism of phage T4 origin-dependent DNA replication.
In vivo assays have provided strong evidence that
transcripts from the origin promoter persist in a
stable RNA-DNA hybrid (R loop). Both in vivo and in
vitro experiments reveal that the RNA within the stable
R loop is used as a primer for leading-strand DNA
replication from the origin.
A second distinct mechanism of replication initiation
in T4 requires phage-encoded recombination proteins.
We have shown that this recombination-dependent replication
(RDR) can be triggered by double-strand breaks in
the genome, and we are analyzing the mechanistic details
of this reaction. RDR involves the formation of a
D-loop, with the invading 3’ end of the DNA
being used as a primer for the leading-strand replication.
RDR has recently been discovered to be an important
mechanism that repairs broken replication forks in
all cells, and disturbances in this process can lead
to genome instability.
Much interest has recently focused on the physiological
roles of DNA helicases, particularly with the discovery
of a helicase defect in human Bloom’s and Werner’s
syndromes, which cause cancer predisposition. We have
shown that the phage T4 UvsW protein is a helicase
that inhibits origin replication, by unwinding the
origin R-loops, and promotes RDR, presumably by its
action on recombination intermediates. We are investigating
the precise function of UvsW helicase in phage recombination,
repair and replication fork restart.
We are also interested in the mechanism of cytotoxicity
of inhibitors of type II DNA topoisomerases. These
inhibitors include important anticancer agents, such
as doxorubicin and etoposide, and the fluoroquinolone
group of antibacterial agents, such as ciprofloxacin.
All of these inhibitors stabilize a reaction intermediate,
called the cleavage complex, in which the topoisomerase
is covalently attached to cleaved DNA. Evidence from
a variety of systems demonstrates that formation of
the cleavage complex is necessary for cytotoxicity,
but is not sufficient. Instead, overt DNA breaks are
apparently formed by an unknown mechanism. We are
attempting to understand the mechanism of break formation
with both the anticancer drugs and the antibacterial
drugs. We have shown that drug-stabilized cleavage
complexes block replication forks in vivo, and have
obtained evidence for a new “collateral damage”
model of drug cytotoxicity. In this model, drug-stabilized
cleavage complexes block the replication fork, and
this blocked fork is later cleaved by recombination
nucleases.
Program trainees play key roles in all of the research
projects within the lab, with each student carrying
one key aspect of the research on their own.
