The central region of RepE initiator protein of mini-F plasmid plays a crucial role in dimerization required for negative replication control

Matsunaga, Fujihiko; Ishiai, Masamichi; Kobayashi, Gengo; Uga, Hisatoshi; Yura, Takashi; Wada, Chieko

doi:10.1006/jmbi.1997.1373

Cited by 30 publications

(26 citation statements)

References 26 publications

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“…5). It should be noted that published work has shown that the WT RepE is dimeric, whereas the mutant form is monomeric (14). It is possible that the lower efficiency and much higher concentrations of the WT protein are needed to catalyze replication of the template reflects residual monomers present in the majority of dimers in the population of WT RepE.…”

Section: Proteins Used In the Inmentioning

confidence: 98%

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Reconstitution of F Factor DNA Replication in Vitro with Purified Proteins

Zzaman

Abhyankar

Bastia

2004

Journal of Biological Chemistry

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Jacob, Brenner, and Cuzin pioneered the development of the F plasmid as a model system to study replication control, and these investigations led to the development of the "replicon model" (Jacob, F., Brenner, S., and Cuzin, F. (1964) Cold Spring Harbor Symp. Quant. Biol. 28, 329 -348). To elucidate further the mechanism of initiation of replication of this plasmid and its control, we have reconstituted its replication in vitro with 21 purified host-encoded proteins and the plasmid-encoded initiator RepE. The replication in vitro was specifically initiated at the F ori (oriV) and required both the bacterial initiator protein DnaA and the plasmidencoded initiator RepE. The wild type dimeric RepE was inactive in catalyzing replication, whereas a monomeric mutant form called RepE* (R118P) was capable of catalyzing vigorous replication. The replication topology was mostly of the Cairns form, and the fork movement was unidirectional and mostly from right to left. The replication was dependent on the HU protein, and the structurally and functionally related DNA bending protein IHF could not efficiently substitute for HU. The priming was dependent on DnaG primase. Many of the characteristics of the in vitro replication closely mimicked those of in vivo replication. We believe that the in vitro system should be very useful in unraveling the mechanism of replication initiation and its control.

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Section: Proteins Used In the Inmentioning

confidence: 98%

“…Mutations in the region of the amino acid residues 111-161 cause a high copy number and the concomitant monomerization of the protein (14). The mini F plasmid is not maintained in host cells that are deficient in DnaK, DnaJ, and GrpE proteins.…”

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Reconstitution of F Factor DNA Replication in Vitro with Purified Proteins

Zzaman

Abhyankar

Bastia

2004

Journal of Biological Chemistry

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“…RepE mutants that are defective in dimerization have also been reported, one of which was used in the crystallization studies (15,22,37). These mutations map to a region of RepE where high copy mutations map (38,39).…”

Section: Molecular Modeling Of P1mentioning

confidence: 99%

Plasmid P1 RepA Is Homologous to the F Plasmid RepE Class of Initiators

Sharma

Sathyanarayana

Bird

et al. 2004

Journal of Biological Chemistry

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DNA replication of plasmid P1 requires a plasmidencoded origin DNA-binding protein, RepA. RepA is an inactive dimer and is converted by molecular chaperones into an active monomer that binds RepA binding sites. Although the sequence of RepA is not homologous to that of F plasmid RepE, we found by using fold-recognition programs that RepA shares structural homology with RepE and built a model based on the RepE crystal structure. We constructed mutants in the two predicted DNA binding domains to test the model. As expected, the mutants were defective in P1 DNA binding. The model predicted that RepA binds the first half of the binding site through interactions with the C-terminal DNA binding domain and the second half through interactions with the N-terminal domain. The experiments supported the prediction. The model was further supported by the observation that mutants defective in dimerization map to the predicted subunit interface region, based on the crystal structure of pPS10 RepA, a RepE family member. These results suggest P1 RepA is structurally homologous to plasmid initiators, including those of F, R6K, pSC101, pCU1, pPS10, pFA3, pGSH500, Rts1, RepHI1B, RepFIB, and RSF1010.

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“…Negative regulation of replication is also given by an interaction between the ori2 and incC iterons mediated by RepE proteins (8), in which a RepE-bridged DNA loop is formed (6). Both initiator and repressor activities, including the interaction between RepE and DNA, have been discussed using several mutants (3,(9)(10)(11)(12)(13)(14), and the first crystal structure of the prokaryotic replication initiator RepE54 [a stable monomeric mutant R118P of RepE isolated from a dnaJ-defective host (10)] in complex with an iteron DNA (RepE54-iteron) has been revealed ( Fig. 1D) (15).…”

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Structural basis for regulation of bifunctional roles in replication initiator protein

Nakamura

Wada

Miki

2007

Proc. Natl. Acad. Sci. U.S.A.

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DNA replication initiator protein RepE stringently regulates F plasmid replication by its two distinct molecular association states. A predominant dimer functions as an autogenous repressor, whereas monomers act as replication initiators, and the dimer requires actions of the DnaK molecular chaperone system for monomerization. The structure of the monomeric form is known, whereas the dimeric structure and structural details of the dimerto-monomer conversion have been unclear. Here we present the crystal structure of the RepE dimer in complex with the repE operator DNA. The dimerization interface is mainly formed by intermolecular ␤-sheets with several key interactions of charged residues. The conformations of the internal N-and C-terminal domains are conserved between the dimer and monomer, whereas the relative domain orientations are strikingly different, allowing for an efficient oligomeric transition of dual-functional RepE. This domain relocation accompanies secondary structural changes in the linker connecting the two domains, and the linker is included in plausible DnaK/DnaJ-binding regions. These findings suggest an activation mechanism for F plasmid replication by RepE monomerization, which is induced and mediated by the DnaK system. DnaK ͉ dual function ͉ F plasmid ͉ RepE ͉ repressor-operator complex

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The central region of RepE initiator protein of mini-F plasmid plays a crucial role in dimerization required for negative replication control

Cited by 30 publications

References 26 publications

Reconstitution of F Factor DNA Replication in Vitro with Purified Proteins

Reconstitution of F Factor DNA Replication in Vitro with Purified Proteins

Plasmid P1 RepA Is Homologous to the F Plasmid RepE Class of Initiators

Structural basis for regulation of bifunctional roles in replication initiator protein

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