Mechanism of DNA binding by the DnaB helicase of Escherichia coli: analysis of the roles of domain gamma in DNA binding.

We have analyzed the mechanism of single-stranded DNA (ssDNA) binding mediated by the C-terminal domain gamma of the DnaB helicase of Escherichia coli. Sequence analysis of this domain indicated a specific basic region, "RSRARR", and a leucine zipper motif that are likely involved in ssDNA binding. We have carried out deletion as well as in vitro mutagenesis of specific amino acid residues in this region in order to determine their function(s) in DNA binding. The functions of the RSRARR domain in DNA binding were analyzed by site-directed mutagenesis. DnaBMut1, with mutations R(328)A and R(329)A, had a significant decrease in the DNA dependence of ATPase activity and lost its DNA helicase activity completely, indicating the important roles of these residues in DNA binding and helicase activities. DnaBMut2, with mutations R(324)A and R(326)A, had significantly attenuated DNA binding as well as DNA-dependent ATPase and DNA helicase activities, indicating that these residues also play a role in DNA binding and helicase activities. The role(s) of the leucine zipper dimerization motif was (were) determined by deletion analysis. The DnaB Delta 1 mutant with a 55 amino acid C-terminal deletion, which left the leucine zipper and basic DNA binding regions intact, retained DNA binding as well as DNA helicase activities. However, the DnaB Delta 2 mutant with a 113 amino acid C-terminal deletion that included the leucine zipper dimerization motif, but not the RSRARR sequence, lost DNA binding, DNA helicase activities, and hexamer formation. The major findings of this study are (i) the leucine zipper dimerization domain, I(361)-L(389), is absolutely required for (a) dimerization and (b) ssDNA binding; (ii) the base-rich RSRARR sequence is required for DNA binding; (iii) three regions of domain gamma (gamma I, gamma II, and gamma III) differentially regulate the ATPase activity; (iv) there are likely three ssDNA binding sites per hexamer; and (v) a working model of DNA unwinding by the DnaB hexamer is proposed.     

Quantitative analysis of binding of single-stranded DNA by Escherichia coli DnaB helicase and the DnaB x DnaC complex

DnaB helicase is responsible for unwinding duplex DNA during chromosomal DNA replication and is an essential component of the DNA replication apparatus in Escherichia coli. We have analyzed the mechanism of binding of single-stranded DNA (ssDNA) by the DnaB x DnaC complex and DnaB helicase. Binding of ssDNA to DnaB helicase was significantly modulated by nucleotide cofactors, and the modulation was distinctly different for its complex with DnaC. DnaB helicase bound ssDNA with a high affinity [Kd = (5.09 +/- 0.32) x 10(-8) M] only in the presence of ATPgammaS, a nonhydrolyzable analogue of ATP, but not other nucleotides. The binding was sensitive to ionic strength but not to changes in temperature in the range of 30-37 degrees C. On the other hand, ssDNA binding in the presence of ADP was weaker than that observed with ATPgammaS, and the binding was insensitive to ionic strength. DnaC protein hexamerizes to form a 1:1 complex with the DnaB hexamer and loads it onto the ssDNA by forming a DnaB6 x DnaC6 dodecameric complex. Our results demonstrate that the DnaB6 x DnaC6 complex bound ssDNA with a high affinity [Kd = (6.26 +/- 0.65) x 10(-8) M] in the presence of ATP, unlike the DnaB hexamer. In the presence of ATPgammaS or ADP, binding of ssDNA by the DnaB6 x DnaC6 complex was a lower-affinity process. In summary, our results suggest that in the presence of ATP in vivo, the DnaB6 x DnaC6 complex should be more efficient in binding DNA as well as in loading DnaB onto the ssDNA than DnaB helicase itself.     

DNA binding and helicase domains of the Escherichia coli recombination protein RecG.

The Escherichia coli RecG protein is a unique junction-specific helicase involved in DNA repair and recombination. The C-terminus of RecG contains motifs conserved throughout a wide range of DNA and RNA helicases and it is thought that this C-terminal half of RecG contains the helicase active site. However, the regions of RecG which confer junction DNA specificity are unknown. To begin to assign structure-function relationships within RecG, a series of N- and C-terminal deletions have been engineered into the protein, together with an N-terminal histidine tag fusion peptide for purification purposes. Junction DNA binding, unwinding and ATP hydrolysis were disrupted by mutagenesis of the N-terminus. In contrast, C-terminal deletions moderately reduced junction DNA binding but almost abolished unwinding. These data suggest that the C-terminus does contain the helicase active site whereas the N-terminus confers junction DNA specificity.     

Multiple-step kinetic mechanism of DNA-independent ATP binding and hydrolysis by Escherichia coli replicative helicase DnaB protein: quantitative analysis using the rapid quench-flow method

The kinetic mechanism of DNA-independent binding and hydrolysis of ATP by the E. coli replicative helicase DnaB protein has been quantitatively examined using the rapid quench-flow technique. Single-turnover studies of ATP hydrolysis, in a non-interacting active site of the helicase, indicate that bimolecular association of ATP with the site is followed by the reversible hydrolysis of nucleotide triphosphate and subsequent conformational transition of the enzyme-product complex. The simplest mechanism, which describes the data, is a three-step sequential process defined by:?eqalign???rm Helicase+ATP?&?mathop??rightleftharpoons? ?k_1?_?k_?-1????rm (H-ATP)??mathop??rightleftharpoons? ?k_2?_?k_?-2????rm (H-ADP?cdot Pi)??cr &?mathop??rightleftharpoons? ?k_3?_?k_?-3????rm (H-ADP?cdot Pi)? *?The sequential character of the mechanism excludes conformational transitions of the DnaB helicase prior to ATP binding. Analysis of relaxation times and amplitudes of the reaction allowed us to estimate all rate and equilibrium constants of partial steps of the proposed mechanism. The intrinsic binding constant for the formation of the (H-ATP) complex is K(ATP)=(1.3+/-0.5)x10(5) M(-1). The analysis of the data indicates that a part of the ATP binding energy originates from induced structural changes of the DnaB protein-ATP complex prior to ATP hydrolysis. The equilibrium constant of the chemical interconversion is K(H)=k(2)/k(-2) approximately 2 while the subsequent conformational transition is characterized by K(3)=k(3)/k(-3) approximately 30. The low value of K(H) and the presence of the subsequent energetically favorable conformational step(s) strongly suggest that free energy is released from the enzyme-product complex in the conformational transitions following the chemical step and before the product release.The combined application of single and multiple-turnover approaches show that all six nucleotide-binding sites of the DnaB hexamer are active ATPase sites. Binding of ATP to the DnaB hexamer is characterized by the negative cooperativity parameter sigma=0.25(+/-0.1). The negative cooperative interactions predominantly affect the ground state of the enzyme-ATP complex. The significance of these results for the mechanism of the free energy transduction of the DnaB helicase is discussed.     

ATP hydrolysis and DNA binding by the Escherichia coli RecF protein.

The Escherichia coli RecF protein possesses a weak ATP hydrolytic activity. ATP hydrolysis leads to RecF dissociation from double-stranded (ds)DNA. The RecF protein is subject to precipitation and an accompanying inactivation in vitro when not bound to DNA. A mutant RecF protein that can bind but cannot hydrolyze ATP (RecF K36R) does not readily dissociate from dsDNA in the presence of ATP. This is in contrast to the limited dsDNA binding observed for wild-type RecF protein in the presence of ATP but is similar to dsDNA binding by wild-type RecF binding in the presence of the nonhydrolyzable ATP analog, adenosine 5'-O-(3-thio)triphosphate (ATPgammaS). In addition, wild-type RecF protein binds tightly to dsDNA in the presence of ATP at low pH where its ATPase activity is blocked. A transfer of RecF protein from labeled to unlabeled dsDNA is observed in the presence of ATP but not ATPgammaS. The transfer is slowed considerably when the RecR protein is also present. In competition experiments, RecF protein appears to bind at random locations on dsDNA and exhibits no special affinity for single strand/double strand junctions when bound to gapped DNA. Possible roles for the ATPase activity of RecF in the regulation of recombinational DNA repair are discussed.     

The interplay of DNA binding, ATP hydrolysis and helicase activities of the archaeal MCM helicase

The MCM (minichromosome maintenance) proteins of archaea are widely believed to be the replicative DNA helicase of these organisms. Most archaea possess a single MCM orthologue that forms homo-multimeric assemblies with a single hexamer believed to be the active form. In the present study we characterize the roles of highly conserved residues in the ATPase domain of the MCM of the hyperthermophilic archaeon Sulfolobus solfataricus. Our results identify a potential conduit for communicating DNA-binding information to the ATPase active site.     

Escherichia coli PriA protein, two modes of DNA binding and activation of ATP hydrolysis

Escherichia coli PriA protein plays crucial roles in processing of arrested replication forks. PriA serves as a sensor/stabilizer for an arrested replication fork and eventually promotes restart of DNA replication through assembly of a primosome. PriA carries a 3' terminus binding pocket required for its high affinity binding to a specific arrested fork as well as for its biological functions. We show here that PriA binds to DNA in a manner either dependent on or independent of 3' terminus recognition. The former mode of binding requires the 3' terminus binding pocket present at the N-terminal half of the 181-residue DNA binding domain and exhibits specific bipartite interaction on the template DNA. The latter mode is independent of the pocket function, but requires the C-terminal half of the same domain. ATP hydrolysis activity of PriA can be stimulated in vitro by either of the two binding modes. We propose architecture of PriA bound to various arrested replication fork structures and discuss its implication in helicase activation and ATP hydrolysis.     

DnaB helicase affects the initiation specificity of Escherichia coli primase on single-stranded DNA templates

The effect of DnaB helicase on the initiation specificity of primase was studied biochemically using a series of single-stranded DNA templates in which each nucleotide of the trinucleotide d(CTG) initiation sequence was systematically varied. DnaB helicase accelerated the rate of primer syntheisis, prevented "overlong" primers from forming and decreased the initiation specificity of primase. In the presence of DnaB helicase, all trinucleotides could serve as the primer initiation site although there was a distinct preference for d(CAG). These data may explain the high chromosomal prevalence of octanucleotides containing CTG on the leading strand and its complement CAG on the lagging strand. The specificity of DnaB helicase places it on the lagging strand template where it stimulates the initiation of Okazaki fragment synthesis. In the absence of DnaB helicase, primase preferentially primed the d(CTG) template. In the presence of DnaB helicase, the initiation preference was not only altered but also the preferred initiating nucleotide was found to be GTP rather than ATP, for both the d(CTG) and the d(CAG) templates. This suggested that the specificity of primase for the d(CTG) initiation trinucleotide was predominantly unaffected in the absence of DnaB helicase on short ssDNA templates, whereas in conjunction with DnaB helicase, the specificity was altered and this alteration has significant implications in the replication of Escherichia coli chromosome in vivo.     

Role of DnaB helicase in UV-induced illegitimate recombination in Escherichia coli

To study the involvement of DNA replication in UV-induced illegitimate recombination, we examined the effect of temperature-sensitive dnaB mutations on illegitimate recombination and found that the frequency of illegitimate recombination was reduced by an elongation-deficient mutation, dnaB14, but not by an initiation-deficient mutation, dnaB252. This result indicates that DNA replication is required for UV-induced illegitimate recombination. In addition, the dnaB14 mutation also affected spontaneous or UV-induced illegitimate recombination enhanced by the recQ mutation. Nucleotide sequence analyses of the recombination junctions showed that DnaB-mediated illegitimate recombination is short homology dependent. Previously, Michel et al. (B. Michel, S. Ehrlich, and M. Uzest, EMBO J. 16:430--438, 1997) showed that thermal treatment of the temperature-sensitive dnaB8 mutant induces double-stranded breaks, implying that induction of illegitimate recombination occurs. To explain the discrepancy between the observations, we propose a model for DnaB function, in which the dnaB mutations may exhibit two types of responses, early and late responses, for double-stranded break formation. In the early response, replication forks stall at damaged DNA, resulting in the formation of double-stranded breaks, and the dnaB14 mutation reduces the double-stranded breaks shortly after temperature shift-up. On the other hand, in the late response, the arrested replication forks mediated by the dnaB8 mutation may induce double-stranded breaks after prolonged incubation.     

Structure-function analysis of Escherichia coli DNA helicase I reveals non-overlapping transesterase and helicase domains

TraI (DNA helicase I) is an Escherichia coli F plasmid-encoded protein required for bacterial conjugative DNA transfer. The protein is a sequence-specific DNA transesterase that provides the site- and strand-specific nick required to initiate DNA strand transfer and a 5' to 3' DNA helicase that unwinds the F plasmid to provide the single-stranded DNA that is transferred from donor to recipient. Sequence comparisons with other transesterases and helicases suggest that these activities reside in the N- and C-terminal regions of TraI, respectively. Computer-assisted secondary structure probability analysis identified a potential interdomain region spanning residues 304-309. Proteins encoded by segments of traI, whose N or C terminus either flanked or coincided with this region, were purified and assessed for catalytic activity. Amino acids 1-306 contain the transesterase activity, whereas amino acids 309-1504 contain the helicase activity. The C-terminal 252 amino acids of the 1756-amino acid TraI protein are not required for either helicase or transesterase activity. Protein and nucleic acid sequence similarity searches indicate that the occurrence of both transesterase- and helicase-associated motifs in a conjugative DNA transfer initiator protein is rare. Only two examples (other than R100 plasmid TraI) were found: R388 plasmid TrwC and R46 plasmid (pKM101) TraH, belonging to the IncW and IncN groups of broad host range conjugative plasmids, respectively. The most significant structural difference between these proteins and TraI is that TraI contains an additional region of approximately 650 residues between the transesterase domain and the helicase-associated motifs. This region is required for helicase activity.     

Interactions of Escherichia coli primary replicative helicase DnaB protein with nucleotide cofactors.

Interactions between the Escherichia coli primary replicative helicase DnaB protein and nucleotide cofactors have been studied using several fluorescent nucleotide analogs and unmodified nucleotides. The thermodynamically rigorous fluorescent titration technique has been used to obtain true binding isotherms, independently of the assumptions of any relationships between the observed quenching of protein fluorescence and the degree of nucleotide binding. Fluorescence titrations using several MANT derivatives of nucleoside diphosphates (MANT-ADP, 3',2'-O-(N-methylantraniloyl)adenosine-5'-diphosphate; MANT-GDP, 3',2'-O(N-methylantraniloyl)guanosine-5'-diphosphate; MANT-CDP, 3',2'-O-(N-methylantraniloyl)cytidine-5'-diphosphate; MANT-UDP, 3',2'-O-(N-methylantraniloyl)uridine-5'-diphosphate) have shown that the DnaB helicase has a preference for purine nucleotides. Binding of all modified nucleotides is characterized by similar negative cooperativity, indicating that negative cooperative interactions are base-independent. Thermodynamic parameters for the interactions of the unmodified nucleotides (ADP, GDP, CDP, and UDP) and inorganic phosphate (P(i)) have been obtained by using the competition titration approach. To analyze multiple ligand binding to a finite circular lattice, for a general case in which each lattice binding site can exist in different multiple states, we developed a matrix method approach to derive analytical expressions for the partition function and the average degree of binding for such cases. Application of the theory to competition titrations has allowed us to extract the intrinsic binding constants and cooperativity parameters for all unmodified ligands. This is the first quantitative estimate of affinities and the mechanisms of binding of different unmodified nucleotides and inorganic phosphate for a hexameric helicase. The intrinsic affinities of all of the studied ATP analogs are lower than the intrinsic affinities of the corresponding ADP analogs. The implications of these results for the mechanism of helicase action are discussed.     

Requirements for ATP binding and hydrolysis in RecA function in Escherichia coli

RecA is essential for recombination, DNA repair and SOS induction in Escherichia coli . ATP hydrolysis is known to be important for RecA's roles in recombination and DNA repair. In vitro reactions modelling SOS induction minimally require ssDNA and non-hydrolyzable ATP analogues. This predicts that ATP hydrolysis will not be required for SOS induction in vivo . The requirement of ATP binding and hydrolysis for SOS induction in vivo is tested here through the study of recA4159 (K72A) and recA2201 (K72R). RecA4159 is thought to have reduced affinity for ATP. RecA2201 binds, but does not hydrolyse ATP. Neither mutant was able to induce SOS expression after UV irradiation. RecA2201, unlike RecA4159, could form filaments on DNA and storage structures as measured with RecA–GFP. RecA2201 was able to form hybrid filaments and storage structures and was either recessive or dominant to RecA⁺, depending on the ratio of the two proteins. RecA4159 was unable to enter RecA⁺ filaments on DNA or storage structures and was recessive to RecA⁺. It is concluded that ATP hydrolysis is essential for SOS induction. It is proposed that ATP binding is essential for storage structure formation and ability to interact with other RecA proteins in a filament.     

The DNA binding properties of the Escherichia coli RecQ helicase

The RecQ helicase family is highly conserved from bacteria to men and plays a conserved role in the preservation of genome integrity. Its deficiency in human cells leads to a marked genomic instability that is associated with premature aging and cancer. To determine the thermodynamic parameters for the interaction of Escherichia coli RecQ helicase with DNA, equilibrium binding studies have been performed using the thermodynamic rigorous fluorescence titration technique. Steady-state fluorescence anisotropy measurements of fluorescein-labeled oligonucleotides revealed that RecQ helicase bound to DNA with an apparent binding stoichiometry of 1 protein monomer/10 nucleotides. This stoichiometry was not altered in the presence of AMPPNP (adenosine 5'-(beta,gamma-imido) triphosphate) or ADP. Analyses of RecQ helicase interactions with oligonucleotides of different lengths over a wide range of pH, NaCl, and nucleic acid concentrations indicate that the RecQ helicase has a single strong DNA binding site with an association constant at 25 degrees C of K=6.7 +/- 0.95 x 10(6) M(-1) and a cooperativity parameter of omega=25.5 +/- 1.2. Both single-stranded DNA and double-stranded DNA bind competitively to the same site. The intrinsic affinities are salt-dependent, and the formation of DNA-helicase complex is accompanied by a net release of 3-4 ions. Allosteric effects of nucleotide cofactors on RecQ binding to DNA were observed only for single-stranded DNA in the presence of 1.5 mM AMPPNP, whereas both AMPPNP and ADP had no detectable effect on double-stranded DNA binding over a large range of nucleotide cofactor concentrations.     

Mechanism and stoichiometry of interaction of DnaG primase with DnaB helicase of Escherichia coli in RNA primer synthesis

Initiation and synthesis of RNA primers in the lagging strand of the replication fork in Escherichia coli requires the replicative DnaB helicase and the DNA primase, the DnaG gene product. In addition, the physical interaction between these two replication enzymes appears to play a role in the initiation of chromosomal DNA replication. In vitro, DnaB helicase stimulates primase to synthesize primers on single-stranded (ss) oligonucleotide templates. Earlier studies hypothesized that multiple primase molecules interact with each DnaB hexamer and single-stranded DNA. We have examined this hypothesis and determined the exact stoichiometry of primase to DnaB hexamer. We have also demonstrated that ssDNA binding activity of the DnaB helicase is necessary for directing the primase to the initiator trinucleotide and synthesis of 11-20-nucleotide long primers. Although, association of these two enzymes determines the extent and rate of synthesis of the RNA primers in vitro, direct evidence of the formation of primase-DnaB complex has remained elusive in E. coli due to the transient nature of their interaction. Therefore, we stabilized this complex using a chemical cross-linker and carried out a stoichiometric analysis of this complex by gel filtration. This allowed us to demonstrate that the primase-helicase complex of E. coli is comprised of three molecules of primase bound to one DnaB hexamer. Fluorescence anisotropy studies of the interaction of DnaB with primase, labeled with the fluorescent probe Ru(bipy)3, and Scatchard analysis further supported this conclusion. The addition of DnaC protein, leading to the formation of the DnaB-DnaC complex, to the simple priming system resulted in the synthesis of shorter primers. Therefore, interactions of the DnaB-primase complex with other replication factors might be critical for determining the physiological length of the RNA primers in vivo and the overall kinetics of primer synthesis.     

Evidence for ATP binding and double-stranded DNA binding by Escherichia coli RecF protein.

RecF protein is one of the important proteins involved in DNA recombination and repair. RecF protein has been shown to bind single-stranded DNA (ssDNA) in the absence of ATP (T. J. Griffin IV and R. D. Kolodner, J. Bacteriol. 172:6291-6299, 1990; M. V. V. S. Madiraju and A. J. Clark, Nucleic Acids Res. 19:6295-6300, 1991). In the present study, using 8-azido-ATP, a photo-affinity analog of ATP, we show that RecF protein binds ATP and that the binding is specific in the presence of DNA. 8-Azido-ATP photo-cross-linking is stimulated in the presence of DNA (both ssDNA and double-stranded DNA [dsDNA]), suggesting that DNA enhances the affinity of RecF protein for ATP. These data suggest that RecF protein possesses independent ATP- and DNA-binding sites. Further, we find that stable RecF protein-dsDNA complexes are obtained in the presence of ATP or ATP-gamma-S [adenosine-5'-O-(3-thio-triphosphate)]. No other nucleoside triphosphates served as necessary cofactors for dsDNA binding, indicating that RecF is an ATP-dependent dsDNA-binding protein. Since a mutation in a putative phosphate-binding motif of RecF protein results in a recF mutant phenotype (S. J. Sandler, B. Chackerian, J. T. Li, and A. J. Clark, Nucleic Acids Res. 20:839-845, 1992), we suggest on the basis of our data that the interactions of RecF protein with ATP, with dsDNA, or with both are physiologically important for understanding RecF protein function in vivo.     

ATP hydrolysis during SOS induction in Escherichia coli.

Changes in cellular ATP concentration during SOS induction in strains of Escherichia coli with different levels of RecA and LexA proteins were studied. UV irradiation of RecA+ strains induced a twofold increase in the ATP concentration around the first 20 min, followed by a decrease to the values of nonirradiated cells. On the other hand, mutants defective in RecA protein or with either deficient RecA protease activity or cleavage-resistant LexA repressor did not show any decrease, suggesting that ATP consumption is related to LexA repressor hydrolysis. Furthermore, strains presenting a constitutive synthesis of RecA protein showed the same changes in ATP concentration as the wild-type strain. Likewise, the presence in a RecA+ strain of a LexA(Def) protein, which is defective in its capacity for binding specifically to SOS operators, did not disturb the changes in ATP when compared with the LexA+ RecA+ strain. Moreover, after UV irradiation, a LexA(Def) RecA- double mutant showed an important increase in ATP concentration, which remained elevated for at least 120 min after UV treatment.     

Modulation of enzymatic activities of Escherichia coli DnaB helicase by single-stranded DNA-binding proteins

The modulation of enzymatic activities of Escherichia coli DnaB helicase by homologous and heterologous single-stranded DNA-binding proteins (SSBs) and its DNA substrates were analyzed. Although DnaB helicase can unwind a variety of DNA substrates possessing different fork-like structures, the rate of DNA unwinding was significantly diminished with substrates lacking a 3′ fork. A 5 nt fork appeared to be adequate to attain the maximum rate of DNA unwinding. Efficient helicase action of DnaB requires the participation of SSBs. Studies involving heterologous SSBs demonstrated that they can stimulate the helicase activity of DnaB protein under certain conditions. However, this stimulation occurs in a manner distinctly different from that observed with cognate E.coli SSB. The E.coli SSB was found to stimulate the helicase activity over a wide range of SSB concentrations and was unique in its strong inhibition of single-stranded DNA-dependent ATPase activity when uncoupled from the DNA helicase activity. In the presence of a helicase substrate, the ATPase activity of DnaB helicase remained uninhibited. Thus, E.coli SSB appears to coordinate and couple the ATPase activity to the DNA helicase activity by suppressing unproductive ATP hydrolysis by DnaB helicase.     

Escherichia coli replicative helicase PriA protein-single-stranded DNA complex. Stoichiometries, free energy of binding, and cooperativities

Analyses of interactions of the Escherichia coli replicative helicase, PriA protein, with a single-stranded (ss) DNA have been performed, using the quantitative fluorescence titration technique. The stoichiometry of the PriA helicase.ssDNA complex has been examined in binding experiments with a series of ssDNA oligomers. The total site-size of the PriA.ssDNA complex, i.e. the maximum number of nucleotide residues occluded by the PriA helicase in the complex, is 20 +/- 3 residues per protein monomer. However, the protein can efficiently form a complex with a minimum of 8 nucleotides. Thus, the enzyme has a strong ssDNA-binding site that engages in direct interactions with a significantly smaller number of nucleotides than the total site-size. The ssDNA-binding site is located in the center of the enzyme molecule, with the protein matrix protruding over a distance of approximately 6 nucleotides on both sides of the binding site. The analysis of the binding of two PriA molecules to long oligomers was performed using statistical thermodynamic models that take into account the overlap of potential binding sites, cooperative interactions, and the protein.ssDNA complexes with different stoichiometries. The intrinsic affinity depends little upon the length of the ssDNA. Moreover, the binding is accompanied by weak cooperative interactions.     

The interaction of bacteriophage P2 B protein with Escherichia coli DnaB helicase

Bacteriophage P2 requires several host proteins for lytic replication, including helicase DnaB but not the helicase loader, DnaC. Some genetic studies have suggested that the loading is done by a phage-encoded protein, P2 B. However, a P2 minichromosome containing only the P2 initiator gene A and a marker gene can be established as a plasmid without requiring the P2 B gene. Here we demonstrate that P2 B associates with DnaB. This was done by using the yeast two-hybrid system in vivo and was confirmed in vitro, where (35)S-labeled P2 B bound specifically to DnaB adsorbed to Q Sepharose beads and monoclonal antibodies directed against the His-tagged P2 B protein were shown to coprecipitate the DnaB protein. Finally, P2 B was shown to stabilize the opening of a reporter origin, a reaction that is facilitated by the inactivation of DnaB. In this respect, P2 B was comparable to lambda P protein, which is known to be capable of binding and inactivating the helicase while acting as a helicase loader. Even though P2 B has little similarity to other known or predicted helicase loaders, we suggest that P2 B is required for efficient loading of DnaB and that this role, although dispensable for P2 plasmid replication, becomes essential for P2 lytic replication.     

Fine balance in the regulation of DnaB helicase by DnaC protein in replication in Escherichia coli.

The DnaC protein of Escherichia coli is essential for replication in vivo and in vitro. In the initiation of replication of a minichromosome at its origin, DnaC delivers the DnaB helicase from a DnaB.DnaC complex to the future replication fork and then departs. However, if an excess of DnaC was present in subsequent steps, it severely inhibited replication by slowing the DnaB helicase at the replication fork. When DnaB was present at a level equimolar with the excess DnaC, the inhibition was relieved, implying that the ratio of DnaC to DnaB is critical for achieving optimal replication activity and avoiding inhibition by DnaC. In vivo, overproduction of DnaC slowed cell growth. This slowing was alleviated by overproducing DnaB at the same time. E. coli strains with a dnaCts gene defective in chromosomal initiation were complemented by the wild-type gene in trans. On the other hand, strains with an elongation-defective dnaCts gene were not complemented by the wild-type dnaC gene. The dominance of the mutant protein suggests that it remains tightly complexed with DnaB at the replication fork, inhibiting elongation even in the presence of the wild-type DnaC.