Replication and structure of DNA
Our
current research centers around the replication of DNA. We wish to
learn why organisms have evolved a variety of strategies for the
replication of their genes. In many bacteriophages, DNA replication
initiates from a well defined origin, but in some instances the genes
are replicated unidirectionally, whereas in others a bidirectional
mechanism is employed. Some genomes replicate as circular structures
while others are linear. In higher organisms replication initiates from
a large number of positions along the genome. What advantages are
conferred by these various replication strategies?
Another
related area of interest concerns the control of DNA replication. How
does an organism achieve balanced replication so that its genes are
neither under- or over-replicated and how is this coupled with the
actual physical division of the cell? We have recently discovered how
to interfere with the control process that is concerned with the
primary initiation of DNA replication in bacteriophage lambda, and in
this way hope to gain insights into the control process itself. Phage
lambda DNA initiates bidirectional replication from a unique position
on its genome, and once replication has started, two daughter origin
sequences are formed. Normally these daughter origins are inactive
while a round of replication is in progress. Apparently a control
process prevents daughter origins from initiating a new wave of
replication until the current round terminates. Under a number of
abnormal conditions we can disturb this control process. For instance,
if replication takes place in the presence of caffeine or the antitumor
drug cis-Pt, daughter origins can reinitiate while a round of
replication is in progress, leading to multiple waves of replication
and complex replicative structures. We are presently investigating
these aberrant replicators in order to determine how the control
process operates.
During the first round of replication
the bidirectional lambda replicator has a theta configuration; the
parental section is negatively supertwisted while the two daughter
segments are relaxed. The control process, just discussed, may simply
arise from supertwisting. A supertwisted origin sequence might be
active, whereas if it is relaxed (as in the daughter section of the
replicative intermediate) it might be inert. To more fully explore this
possibility we have developed a novel electron microscopic method that
allows us to unambiguously determine the supertwisted state of DNA in
complex replicative structures. The method is based on the fact that
the efficiency of intercalation of the drug psoralen is much higher for
negatively supertwisted than for relaxed DNA.
Our
research relies heavily on physical and electron microscopic methods;
our world wide web page shows a variety of electron micrographs.