The localized melting of mini-F origin by the combined action of the mini-F initiator protein (RepE) and HU and DnaA of Escherichia coli

Abstract

Replication of mini-F plasmids requires the initiator protein RepE, which binds specifically to four iterons within the origin (ori2), as well as some host factors that are involved in chromosomal DNA replication. To understand the role of host factors and RepE in the early steps of mini-F DNA replication, we examined the effects of RepE and the Escherichia coli proteins DnaA and HU on the localized melting of ori2 DNA in a purified in vitro system. We found that the binding of RepE to an iteron causes a 50 degrees bend at or around the site of binding. RepE and HU exhibited synergistic effects on the localized melting within the ori2 region, as detected by sensitivity to the single-strand specific P1 endonuclease. This opening of duplex DNA occurred around the 13mer of ori2, whose sequence closely resembles the set of 13mers found in the chromosomal origin oriC. Further addition of DnaA to the reaction mixture increased the efficiency of melting and appeared to extend melting to the adjacent AT-rich region. Moreover, DNA melting with appreciably higher efficiencies was observed with mutant forms of RepE that were previously shown to be hyperactive both in DNA binding in vitro and in initiator activity in vivo. We propose that the binding of RepE to four iterons of ori2 causes bending at the sites of RepE binding and, with the assistance of HU, induces a localized melting in the 13mer region. The addition of DnaA extends melting to the AT-rich region, which could then serve as the entry site for the DnaB-DnaA complex, much as has been documented for oriC dependent replication.

Role of RepA and DnaA Proteins in the Opening of the Origin of DNA Replication of an IncB Plasmid

“…DnaA appeared to play an auxiliary role in localized unwinding of ori B , augmenting the function of RepA without itself being able to open this origin. In this respect, the role of DnaA in origin opening of ori B was similar to that played by this protein in opening the origins of plasmids RK2 (23) and F (19). However, whereas replication of RK2 and F is absolutely dependent on DnaA (11,21), pMU720 can replicate in a dnaA-null host (Borrell and Praszkier, unpub- .…”

“…However, given that one DnaB hexamer binds a singlestranded region of 20 Ϯ 3 nt (18), it is likely that the region of ori B opening in vivo is larger than that detected in the present study, as host proteins other than DnaA may also contribute to opening of ori B . One such protein, HU, is required for origin opening in plasmids P1 (38) and F (19).…”

“…The AT-rich regions in the origins of replication of several members of the iteron-regulated family of plasmids also contain 13-mers whose sequences resemble those of the 13-mers present in oriC. Thus, pSC101 has two tandem 13-mers matching the consensus of those in oriC (5); F has one 13-mer, which has a single mismatch with the oriC consensus (19); and RK2 has four 13-mers bearing the consensus 5Ј-TAAACnTTnTTTT-3Ј (23). The AT-rich region of ori B of pMU720, which is not an iteron-regulated plasmid but a member of the extended family of FII/I complex group of plasmids, contains a 6-mer 5Ј-TCTTAA-3Ј that is conserved in the origins of replication of plasmids distantly related to pMU720 (2).…”

“…All plasmids showing absolute or partial dependence on DnaA contain one or more copies of the DnaA R box in their origin of replication. Analyses of the role of DnaA in replication of these plasmids revealed that it is different for different plasmids but involves participation in open complex formation, recruitment of DnaB helicase, or both (8,19,23,24,27,37,38,46). pMU720, unlike other theta-replicating plasmids of gramnegative bacteria that have been characterized to date, does not belong to the family of iteron-regulated plasmids, but is a member of the extended family of IncFII-related replicons whose copy number is regulated by an antisense RNA.…”

Cloning and Expression inEscherichia coliof a Gene,hup, Encoding the Histone-like Protein HU ofBifidobacterium longum

“…1) The resulting alteration of DNA structure aŠects the regulation of initiation of DNA replication, [2][3][4] transposition of Mu phage, [5][6][7] cell division, 8) site-speciˆc DNA recombination, 9) and transcription. 10) HU generally exists as a homodimer, although it consists of two distinct HU subunits HUa (encoded from hupA) and HUb (encoded from hupB ) in E. coli, Salmonella typhimurium, and Serratia marcescens.…”