|
B. DEPARTMENT OF
CELL GENETICS
B-b. Division of Microbial Genetics - Seiichi
Yasuda Group
RESEARCH
ACTIVITIES
(1)
Mechanism of DnaA interaction with
phospholipids
Seiichi Yasuda
--The replication
of chromosome in Escherichia coli is
initiated at a unique chromosomal locus,
oriC. A protein called DnaA catalyzes the
first step in the initiation by binding and opening
double-stranded DNA at an AT-rich region in
oriC. The opened single-stranded region
serves as the site of assembly of other proteins to
form a replication complex. Comparative studies on
the dnaA genes of many bacterial strains
have established that DnaA is made of four
functional domains. Among them, domain 3 is
responsible for ATP-binding, and domain 4 for
sequence-specific binding to oriC DNA. It
has been known that acidic phospholipids such as
cardiolipin release bound ATP and ADP from DnaA.
This phenomenon was suggested to be involved in
rejuvenation of used DnaA. It has been postulated
that DnaA interacts with phospholipids at a site
near the C-terminal portion of domain 3 because
this site has an amino acid sequence that can form
an alpha helix having a hydrophobic surface.
--Previous studies
with mutant DnaA proteins in this laboratory showed
that the interaction of DnaA with phospholipids
depends on the presence of the DNA-binding domain
of DnaA, but not on that of the putative
mem-brane-interaction site in domain III. This was
further confirmed by quantitative measurements of
DNA-binding and phospholipid-induced ATP-release
using mutant DnaA proteins. DnaA mutants used were
DnaADelM that has a deletion of the
membrane-interaction site, DnaADelV that has a
deletion of C-terminal 14 amino acids of the
DNA-binding domain, and DnaADelS that has a
deletion of almost all of the DNA-binding domain
but that retains the membrane-interaction site. ATP
binding of all these mutant proteins was
essentially the same as that of wild type DnaA.
Dissociation constants of DNA-binding were measured
and found to be 5.75, 16.5, 115, and 825 nM for
wild type DnaA, DnaADelM, DnaADelV and DnaADelS,
respectively. The rate of ATP release versus
cardiolipin concentration was determined and it was
found that the order of cardiolipin sensitivity of
these DnaA proteins was exactly reverse to that of
the DNA binding constants. This suggests strongly
that cardiolipin interacts with DnaA at the
DNA-binding domain leading to the release of ATP
from the protein.
--During the course of
this work it was found that cardiolipin inactivates
the ATP, and DNA-binding activities of DnaA, but
not the replication activity as measured in the
presence of oriC and a crude protein fraction. The
crude protein fraction was found to reactivate DnaA
that had been inactivated by cardiolipin.
Characterization of the reactivating factor is
under way.
|
