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Abstract
The segregation of many bacterial chromosomes is dependent on the interactions of ParB proteins with centromere-like DNA sequences called parS that are located close to the origin of replication. In this work, we have investigated the binding of Bacillus subtilis ParB to DNA in vitro using a variety of biochemical and biophysical techniques. We observe tight and specific binding of a ParB homodimer to the parS sequence. Binding of ParB to non-specific DNA is more complex and displays apparent positive co-operativity that is associated with the formation of larger, poorly defined, nucleoprotein complexes. Experiments with magnetic tweezers demonstrate that non-specific binding leads to DNA condensation that is reversible by protein unbinding or force. The condensed DNA structure is not well ordered and we infer that it is formed by many looping interactions between neighbouring DNA segments. Consistent with this view, ParB is also able to stabilize writhe in single supercoiled DNA molecules and to bridge segments from two different DNA molecules in trans. The experiments provide no evidence for the promotion of non-specific DNA binding and/or condensation events by the presence of parS sequences. The implications of these observations for chromosome segregation are discussed.
Highlights
ParABS systems were discovered and defined as the DNA segregation apparatus for low copy number plasmids [1]
To investigate binding of ParB to specific and non-specific DNA we first developed a protocol for the purification of native ParB protein and re-visited the use of Electrophoretic mobility shift assays (EMSAs) assays using a variety of different buffer compositions for resolving nucleoprotein complexes in the gels
When TBE buffer was used for the resolving gel as in previous experiments (Supplementary Figure S1) [6,10,12], we found that ParB bound to a 147-bp DNA molecule to form a ladder of shifted bands that was highly reminiscent of those seen before [10,12]
Summary
ParABS systems were discovered and defined as the DNA segregation apparatus for low copy number plasmids [1] They comprise the ParB protein, which binds to the centromere-like DNA sequence parS, as well as ParA; a DNA binding protein and ATPase that binds DNA non- in the presence of adenosine triphosphate (ATP). These three partitioning factors act together to distribute plasmids into daughter cells. Eight functional parS sequences are found in the origin proximal region of the B. subtilis chromosome [6] and ParB has been shown to associate with DNA for several kilobases around each of these sites [11,12]. The mechanism of ParB spreading from parS, along with many other aspects of chromosomal ParABS function, is not fully understood
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