How Do Microbes Reproduce?
|
In prokaryotes, genetic information is contained in the DNA in
a single chromosome and in smaller DNA molecules called plasmids,
but the P1 plasmid par system is a model for partition in both
cases. The ParB–centromere partition complex serves as
the docking site for ParA, the enzyme (ATPase) that drives subsequent
separation of the plasmid DNA. ParB(142-333) contains all the
determinants required for centromere binding and formation of
the partition complex.
A parS-small centromere site with 25 base pairs (bp)
and two DNA structural units (motifs) called the A-Box and B-Box
is the minimal partition site required for segregation. However,
partition efficiency is increased when a full-length, 74-bp parS centromere
is used and the site is bent by the host auxiliary factor IHF whose
role is simply to bring together the parS arms that contain
the A-Box and B-Box. Once the initial complex is formed,
additional ParB molecules load onto and spread along the DNA to
form large nucleoprotein complexes. These findings indicate a highly
complex P1 partition interaction topology and a protein–DNA
interaction between ParB and the centromere that is unlike any
previously described. In this interaction, ParB must, in some unknown
manner, bridge the juxtaposed arms of a looped parS centromere
site.
Of more than 70 samples in crystallization trials, only two provided
data beyond 4.0-Å resolution, and data for these were collected
at ALS Beamlines 8.2.1 and 5.0.2. Two structures of the P1 ParB(142-333)–parS-small partition
complex were determined, the first by multiple-wavelength anomalous
dispersion (MAD) and the second by molecular replacement. Both
structures reveal that ParB forms an asymmetric dimer with two
flexibly linked DNA-binding modules: the extended N-terminal helix-turn-helix
(HTH)- containing domains, which contact A-Boxes, and the novel
dimerized Dimer domain, which contacts B-Box elements.
Crystal structure of the P1 ParB(142-333)–parS-small partition
complex. The asymmetric ParB dimer is shown as a ribbon diagram
with one subunit colored cyan and the other subunit magenta.
Each ParB(142-333) subunit consists of two flexibly attached
domains: the HTH domain, which contacts the A-Box DNA, and the
Dimer domain, which contacts B-Box DNA. The parS-small site
is displayed as a surface representation with the A2-, A3-, and
B2-Boxes colored yellow, green, and blue, respectively. Next
to the structure is the sequence of the parS-small centromere
site.
In fact, the structures in the two crystal forms, which reveal
domain rotations ranging from about 60º to 160º relative
to one another, suggest that the flexible linker between the DNA-binding
modules allows them to rotate essentially freely. Strikingly, such
free rotation would permit these modules to contact direct or inverted
arrangements of A- and B-Boxes of the type found in parS.
Most remarkably, however, each DNA-binding element binds to and
thus bridges adjacent DNA duplexes.
Ribbon diagram showing the four distinct DNA bridging interactions
observed in two different crystal forms of the complex. ParB and
Box motifs are colored as before, and the DNA is rendered as a
transparent surface. ParB is shown in the same orientation in both
crystal forms to underscore the ability of the DNA-binding modules
to undergo essentially free rotation, permitting them to contact
distinct orientations of Box elements on different DNA duplexes.
The composite and flexibly linked DNA-binding modules and the ability
of these flexibly attached elements to bridge adjacent DNA duplexes
are unique for a DNA-binding protein and explain how this protein
can bind complex arrays of A- and B-Box elements on adjacent DNA
arms of the looped centromere site. Moreover, the unique bridging
function of ParB may play a role in mediating plasmid pairing. Plasmid
pairing is the next crucial step in partition after initial formation
of the complex whereby the two DNA molecules are brought together.
The paired complexes are then separated by the action of the ParA
ATPase. How pairing occurs has been unknown, but the structures suggest
that ParB mediates pairing by bridging between the two arms of one parS site
and simultaneously binding to a second plasmid parS site.
Research conducted by M.A. Schumacher (University of Texas, MD
Anderson Cancer Center) and B.E. Funnell (University of Toronto).
Research funding: Burroughs Wellcome Career Development Award
and Canadian Institutes of Health Research. Operation of the ALS
is supported by the U.S. Department of Energy, Office of Basic
Energy Sciences (BES).
Publication about this research: M.A. Schumacher and B.E. Funnell, “Structures
of ParB bound to DNA reveal mechanism of partition complex formation,” Nature 438,
516 (2005).
ALSNews
Vol. 263, March 29, 2006 |