A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis.
Two dimers of the replication terminator protein (RTP) of Bacillus subtilis bind to a chromosomal DNA terminator site to effect polar replication fork arrest. Cooperative binding of the dimers to overlapping half-sites within the terminator is essential for arrest. It was suggested previously that polarity of fork arrest is the result of the RTP dimer at the blocking (proximal) side within the complex binding very tightly and the permissive-side RTP dimer binding relatively weakly. In order to investigate this "differential binding affinity" model, we have constructed a series of mutant terminators that contain half-sites of widely different RTP binding affinities in various combinations. Although there appeared to be a correlation between binding affinity at the proximal half-site and fork arrest efficiency in vivo for some terminators, several deviated significantly from this correlation. Some terminators exhibited greatly reduced binding cooperativity (and therefore have reduced affinity at each half-site) but were highly efficient in fork arrest, whereas one terminator had normal affinity over the proximal half-site, yet had low fork arrest efficiency. The results show clearly that there is no direct correlation between the RTP binding affinity (either within the full complex or at the proximal half-site within the full complex) and the efficiency of replication fork arrest in vivo. Thus, the differential binding affinity over the proximal and distal half-sites cannot be solely responsible for functional polarity of fork arrest. Furthermore, efficient fork arrest relies on features in addition to the tight binding of RTP to terminator DNA.
Duggin
Iain G
IG
School of Molecular and Microbial Biosciences, The University of Sydney, New South Wales 2006 and Research School of Chemistry, Australian National University, Australian Capital Territory 0200, Australia. iduggin@gmail.com
Matthews
Jacqueline M
JM
Dixon
Nicholas E
NE
Wake
R Gerry
RG
Mackay
Joel P
JP
D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D015139:Blotting, Southern; D002151:Calorimetry; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004261:DNA Replication; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D014461:Ultracentrifugation
D004247:DNA