|DNA-Binding Mechanism in Prokaryotic Partition Complex Formation|
The faithful inheritance of genetic information, essential for all organisms, requires accurate movement and positioning of replicated DNA to daughter cells during cell division. In cells without distinct nuclei (prokaryotes), this process, called partition or segregation, is mediated by par systems. The prototype system of prokaryotic partition is the Escherichia coli P1 plasmid par system, which consists of a centromere site (parS) on the plasmid DNA and two proteins, ParA and ParB. The initial formation of the so-called partition complex between ParB and the centromere is a critical step in partition. To understand the DNA-binding mechanism utilized by ParB, Schumacher and Funnell determined crystal structures of the C-terminal region of ParB, known as ParB(142-333), bound to centromere sites.
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.
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.
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).