Abstract
The P1 ParB protein is required for active partition and thus stable inheritance of the plasmid prophage. ParB and the Escherichia coli protein integration host factor (IHF) participate in the assembly of a partition complex at the centromere-like site parS. In this report the role of IHF in the formation of the partition complex has been explored. First, ParB protein was purified for these studies, which revealed that ParB forms a dimer in solution. Next, the IHF binding site was mapped to a 29-base pair region within parS, including the sequence TAACTGACTGTTT (which differs from the IHF consensus in two positions). IHF induced a strong bend in the DNA at its binding site. Versions of parS which have lost or damaged the IHF binding site bound ParB with greatly reduced affinity in vitro and in vivo. Measurements of binding constants showed that IHF increased ParB affinity for the wild-type parS site by about 10,000-fold. Finally, DNA supercoiling improved ParB binding in the presence of IHF but not in its absence. These observations led to the proposal that IHF and superhelicity assist ParB by promoting its precise positioning at parS, a spatial arrangement that results in a high affinity of ParB for parS.
Highlights
The P1 ParB protein is required for active partition factor, or IHF’ (6).It was identified as a factor that stimulated and stable inheritance of the plasmid prophage
ParB protein was puri- tively (10, 11).IHF participates in a variety of processes in fied for these studies, which revealed that ParB forms E. coli, including recombination, replication, and transcripa dimer in solution
The prophage of bacteriophage P1 exists as an autono- this region (6). hmRoeosfu.tsl1cy)h.rrTeophmleiocpastloianmsgme)pid,laacsnomPpd1iydreniqnuumEirbseecsrhieasrnicvhearicaytciolvoleiw(fpo(aorrnrteeitvitiooenwtwssoeye/stemdwca,eiPnrldinrv-eotavyttiipinovetueespugasrsreattdtuietditinooietnostt(ehc6soe)tbmsahppcoaltewerxtreiidtaiinlothnuaiutivoni.IvHoAcFihsilrsoaowamXcco-ooPsmpo1ympcohenn.iumemSneubtroceafhr ttPhhX1ea- t pur, to promote proper plasmid segregation at cell division. miniP1 lysogens exist as P1 plasmids; they require the P1 P1pur encodes two proteins, ParA and ParB, and contaians replication and partition systefmorsproper maintenance(13). centromere-like site,pars, so called because it is required in X-MiniP1 plasmids were less stable in cells without IHF (E. cis for plasmid stability (Fig. 1)(2)
Summary
Vol 266, No 22, Issue of August 5, pp. 14328-14337, 1991 Printed in U.S.A. From the Laboratoryof Biochemistry, National Cancer Institute, National Instituteosf Health, Bethesda, Maryland 20892. Measurementsof binding constants showed that IHF increased ParB affinity for the wild-type pars site by about 10,000-fold. It habseen proposed that coli hip mutants) than in wild-type cells, but the defect was one or both plasmid-encoded proteins bind to pars, which less severethan thatcaused by complete lososf ParB function leads to pairing of plasmids and association of the paired (6) From this it follows that IHF assists ParB but is dispencomplex withthehostpartitionapparatusatthenascent sable whereas ParB is absolutely required. The X-miniPlparS-[Dra] inser- digestion of a 3’ end-labeled EcoRI digest(the EcoRI enwdas formerly tion mutant derivatives were constructed by cloning the insertions the P1 TuqI site; Fig. 1).The 452-bp “upper strand” fragmenrtesulted onto a pBR322 (16) plasmid containing about 2 kb of P1 DNA and from a PstI digest of a 3’ end-labeled HindIII digest of pBEF143.
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