Deepak Bastia

The integration host factor of Escherichia coli binds to bent DNA at the origin of replication of the plasmid pSC101.

The integration host factor (IHF) of Escherichia coli is necessary for maintenance of pSC101. The protein binds specifically to the replication origin of the plasmid, in the AT-rich region located immediately adjacent to the left, weak binding site for the plasmid-encoded initiator protein. DNAase I and OH- radical footprinting experiments showed that IHF protects 49 bp of the DNA at the origin region. Methylation protection analyses revealed that IHF contacts purine residues in both the major and minor grooves of the DNA. Electrophoretic analyses showed that IHF binds to bent DNA, and the protein binding further enhances the degree of DNA bending. Site-directed mutagenesis of three of the contact points not only abolished binding of the protein to the DNA but also inactivated the replication origin. Therefore, binding of IHF to the ori sequence most probably is necessary for initiation of plasmid replication.

The replication terminator protein of E. coli is a DNA sequence-specific contra-helicase

We have cloned the tus gene that encodes the replication terminator protein of Escherichia coli and have efficiently expressed its gene product. The overproducer strain has been used to purify the terminator (ter) protein in high yield to near homogeneity. The protein is a single 36 kd polypeptide. Using the ter protein and highly purified dnaB helicase, we show that the terminator protein is a DNA sequence-specific contra-helicase, i.e., the protein when bound to its recognition sequence (tau) strongly impedes the ATP-dependent unwinding of double-stranded DNA. This contra-helicase activity is polar, i.e., the impedance to unwinding takes place in only one orientation of the tau sequence. The results illuminate the mechanism of replication termination specifically at tau.

Interaction of the plasmid R6K-encoded replication initiator protein with its binding sites on DNA

Initiation of DNA replication in plasmid R6K is potentiated by the plasmid-encoded 35 kd replication initiator protein. We had previously reported that the initiator bound to two regions of R6K DNA called Site I and Site II. Using DNAase I footprinting technique we have demonstrated that the initiator bound to seven tandem repeats of a 22 bp long sequence in Site I. In Site II, the initiator bound to a single repeat having the same consensus sequence and to two partial repeats that most likely overlap the promoter of the initiation protein cistron. Using dimethyl sulfate as a chemical probe, we have determined the purine residues of Site I and Site II that make contact with the initiator protein. The results show that eight out of nine contact points per repeat in Site I were located on one of the two strands of the DNA. The binding of the initiator to the Site II sequence could explain the observed autoregulation of the synthesis of the initiator protein by promoter occlusion.

Termination of DNA replication of bacterial and plasmid chromosomes

Sequence‐specific replication termini occur in many bacterial and plasmid chromosomes and consist of two components: a cis‐acting ter site and a trans‐acting replication terminator protein. The interaction of a terminator protein with the ter site creates a protein–DNA complex that arrests replication forks in a polar fashion by antagonizing the action of the replicative helicase (thereby exhibiting a contrahelicase activity). Terminator proteins also arrest RNA polymerases in a polar fashion. Passage of an RNA transcript through a terminus from the non‐blocking direction abrogates replication termination function, a mechanism that is likely to be used in conditional termini or replication check points.

Mechanistic studies of initiator–initiator interaction and replication initiation

Unlike the chromosome of Escherichia coli that needs only one replication initiator protein (origin recognition protein) called DnaA, many plasmid replicons require dual initiators: host‐encoded DnaA and a plasmid‐encoded origin recognition protein, which is believed to be the major determinant of replication control. Hitherto, the relative mechanistic roles of dual initiators in DNA replication were unclear. Here, we present the first evidence that DnaA communicates with the plasmid‐encoded π initiator of R6K and contacts the latter at a specific N‐terminal region. Without this specific contact, productive unwinding of plasmidori γ and replication is abrogated. The results also show that DnaA performs different roles in host and plasmid replication as revealed by the finding that the ATP‐activated form of DnaA, while indispensable for oriC replication, was not required for R6K replication. We have analyzed the accessory role of the DNA bending protein, integration host factor (IHF), in promoting initiator–origin interaction and have found that IHF significantly enhances the binding of DnaA to its cognate site. Collectively, the results further advance our understanding of replication initiation.

Conformational changes in a replication origin induced by an initiator protein.

The replication initiator protein of the plasmid R6K binds to seven contiguous 22 bp direct repeats that form an indispensable part of the three replication origins alpha, beta, and gamma. Binding of the initiator to the direct repeats induced a marked bending of the region of gamma replication origin. Binding of the initiator also promoted unwinding of the origin DNA by at least two turns. Distamycin appeared to antagonize the binding of the initiator to the seven 22 bp direct repeats. At the appropriate DNA and protein concentrations the initiator enhanced topoisomerase-induced catenation of the origin containing supercoiled DNA but not of DNA lacking the origin sequence. Thus, the initiator protein caused significant changes in the secondary and tertiary structures of the replication origin.

Crystal structure of the replication terminator protein from B. subtiiis at 2.6 Å

The crystal structure of the replication terminator protein (RTP) of B. subtilis has been determined at 2.6 A resolution. As previously suggested by both biochemical and biophysical studies, the molecule exists as a symmetric dimer and is in the alpha + beta protein-folding class. The protein has several uncommon features, including an antiparallel coiled-coil, which serves as the dimerization domain, and both an alpha-helix and a beta-ribbon suitably positioned to interact with the major and minor grooves of B-DNA. A site has been identified on the surface of RTP that is biochemically and positionally suitable for interaction with the replication-specific helicase. Other features of the structure are consistent with the polar contrahelicase mechanism of the protein. A model of the interaction between RTP and its cognate DNA is presented.

Activation of distant replication origins in vivo by DNA looping as revealed by a novel mutant form of an initiator protein defective in cooperativity at a distance

We have isolated mutants of the pi initiator protein of the plasmid R6K that are defective in DNA looping in vitro but retain their normal DNA binding affinity for the primary binding sites (iterons) at the gamma origin/enhancer. One such looping defective mutant called R6 was determined to be a proline to leucine change at position 46 near the N terminus of the pi protein. Using a set of genetic assays that discriminate between the activation of the gamma origin/enhancer from those of the distantly located alpha and beta origins, we show that the looping defective initiator protein fails to activate the alpha and beta origins but derepresses initiation from the normally silent gamma origin in vivo. The results conclusively prove that DNA looping is required to activate distant replication origins located at distances of up to 3 kb from the replication enhancer.

DNA-protein interaction at the origin of DNA replication of the plasmid pSC101

The initiation of DNA replication of the low copy number plasmid pSC101 is dependent on the dnaA initiator protein encoded by Escherichia coli. We have previously reported that the minimum essential replicon of the plasmid encodes a approximately 37.5 kd protein and that the protein is necessary along with host-encoded proteins for the replication of the plasmid chromosome. In this communication we show that the plasmid-encoded protein has sequence-specific DNA-binding activity. The protein binds cooperatively to the replication origin of pSC101. Using chemical and enzymatic probes we have determined the contact points of the protein with the DNA and the precise domain of the replication origin recognized by the 37.5 kd protein. The specificity of the DNA-protein interaction would suggest that the 37.5 kd protein may possibly function by guiding the replisome to the correct DNA sequence on the chromosome of pSC101.

Helicase–Contrahelicase Interaction and the Mechanism of Termination of DNA Replication

Termination of DNA replication at a sequence-specific replication terminus is potentiated by the binding of the replication terminator protein (RTP) to the terminus sequence, causing polar arrest of the replicative helicase (contrahelicase activity). Two alternative models have been proposed to explain the mechanism of replication fork arrest. In the first model, the RTP-terminus DNA interaction simply imposes a polar barrier to helicase movement without involving any specific interaction between the helicase and the terminator proteins. The second model proposes that there is a specific interaction between the two proteins, and that the DNA-protein interaction both restricts the fork arrest to the replication terminus and determines the polarity of the process. The evidence presented in this paper strongly supports the second model.