Mechanism of dnaB protein action. I. Crystallization and properties of dnaB protein, an essential replication protein in Escherichia coli

Purification and crystallization of dnaB protein from Escherichia coli was performed on a large scale by a simple procedure. From 1.5 kg of cells, 520 mg of dnaB protein were obtained in a 58% yield with a purity greater than 99%. The E. coli cells harbor a high copy-number plasmid carrying the dnaB gene and overproduce the enzyme over 200-fold. The subunit molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 50,000. Based on a native Mr = 290,000 and cross-linking studies that yielded six bands, dnaB protein is judged to be a hexamer, confirming the results of Reha-Krantz, L. J., and Hurwitz, J. (1978) J. Biol. Chem. 253, 4043-4050.     

Escherichia coli dnaB protein. Affinity chromatography on immobilized nucleotides.

The purification of the Escherichia coli dnaB protein by affinity chromatography on nucleotides bound to agarose is described. The dnaB protein, which contains an associated ribonucleoside triphosphatase activity (Wickner, S., Wright, M., and Hurwitz, J. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 783-787) binds to immobilized ATP, ADP, and UDP, but not to AMP. The type of linkage of ATP to agarose influences the adsorption, elution, and purification of the enzyme. Optimal purification is achieved using ATP bound to agarose via its oxidized ribose moiety. By this means, the dnaB protein can be obtained at least 95% electrophoretically pure after only three purification steps. The enzyme can be eluted from immobilized nucleoside-5'-di- and -triphosphates by ATP, ADP, and pyrophosphate, but not by AMP or orthophosphate. ADP and pyrophosphate, as well as the substrate ATP in high concentration are at the same time inhibitors of the ribonucleoside triphosphatase. The dnaB complementing and ribonucleoside triphosphatase activities could not be separated from each other by affinity chromatography, supporting the finding of others that they both reside on the same protein complex, namely a dnaB multimer. The results indicate that the dnaB protein binds to immobilized nucleotides by means of its ribonucleoside triphosphatase, and that at least the pyrophosphate moiety is essential for adsorption as well as elution of the enzyme.     

RNA polymerase interaction with dnaB protein and lambda P protein during lambda replication.

The Escherichia coli GroP- phenotype, associated with some dnaB mutants and measured as a decreased ability to plate lambda bacteriophage, was altered by some rpoB mutations. The rpoB effect showed an allele specificity. The participation both of dnaB and of lambda P alleles in the GroP- phenotype was also allele specific. It was concluded that RNA polymerase, dnaB protein, and lambda P protein form a functional complex required for lambda replication.     

The dnaC protein of Escherichia coli. Purification, physical properties and interaction with dnaB protein.

E.coli dnaC protein was purified to near-homogeneity in using a dnaC complementation assay [S.Wickner, I.Berkower, M.Wright, and J.Hurwitz (1973) Proc. Natl. Acad. Sci. USA 70, 2369-2373]. Purification was achieved by taking advantage of the hydrophobic interaction of dnaC protein with aliphatic and aromatic matrixes and with Brij58 as stabilizing agent. A sedimentation coefficient for the dnaC protein of 2.6 S corresponding to a molecular weight of approximately 26,000 was estimated from glycerol gradient centrifugation. A polypeptide molecular weight of 28,000 was determined by densitometry on a denaturing gel. In the presence of ATP the dnaC protein forms a complex with dnaB protein [S.Wickner and J.Hurwitz (1975) Proc.Natl.Acad.Sci. USA 72, 921-925]. For the dnaB . dnaC complex a sedimentation coefficient of 14.5 S was measured by glycerol gradient centrifugation, indicating a molecular weight of about 400,000. The ratio of the dnaC and dnaB polypeptides in the complex is approximately 1, as determined on a denaturing gel. It is suggested that the complex consists of the dnaB protein hexamer and six dnaC polypeptides amounting to a calculated molecular weight of about 450,000.     

Role of dnaB43 in F''-plasmid incompatibility.

Summary In order to perform complementation tests with mutations of DNA replication of F-plasmids incompatibility must be overcome. We report our inability to duplicate the results presented by Palchoudhury and Iyer (1971) and Bezanson and Iyer (1975) who have claimed to demonstrate the autonomous replication of two incompatible F-plasmids in a strain carrying the temperature sensitivednaB43 allele. In addition, we describe experiments designed to measure complementation using transient heterozygote and compatible plasmids. Assessment of our data and those of others in the light of a recent report by Uhlin and Nordström (1975) suggests that new approaches will have to be developed for the successful employment of complementation analysis in F-plasmid genetics.     

Thymineless death in Escherichia coli dnaB mutants and in a dnaB dnaG double mutant.

The interference of dnaB mutations of Escherichia coli with thymineless death is described. All the isogenic Thy- dnaB mutants of E. coli we have tested show a remarkable immunity towards cell death induced by thymine deprivation at the nonpermissive temperature. We have also constructed and tested an isogenic double dnaB dnaG mutant. It loses its viability in the absence of thymine at both permissive and nonpermissive temperatures. The role of the dnaB gene product is discussed.     

dnaB125, a dnaB nonsense mutation

A temperature-sensitive dnaB mutation, dnaB125, was shown to be a suppressed amber mutation. The effects of inserting different amino acids at the mutated site via amber suppressors were examined for both Escherichia coli and bacteriophage gamma growth. In addition, the dnaB125 amber allele was shown to be different from the previously described dnaB amber allele, dnaB266. The extent of residual deoxyribonucleic acid synthesis observed in a supF(Ts) dnaB125 strain at high temperature revealed that the dnaB protein was present in excess and that deoxyribonucleic acid synthesis could continue for several generation equivalents without further production of dnaB protein.     

Suppression of tif-mediated induction of SOS functions in Escherichia coli by an altered dnaB protein.

The tif-1 mutation in the Escherichia coli recA gene is known to cause induction of the various "SOS" functions at high temperature, including massive synthesis of the recA protein, lethal filamentation, elevated mutagenesis, and, in lambda lysogens, induction of prophage. It is shown here that the deoxyribonucleic acid initiation mutation dnaB252 suppresses all these manifestations of tif expression. Induction of lambda by ultraviolet irradiation, however, is not affected by the dnaB252 mutation. No similar suppression of tif is observed with other dnaB mutations affecting deoxyribonucleic acid elongation or with other deoxyribonucleic acid initiation mutations at the dnaA and dnaC loci. The fact that an alteration of the dnaB protein specifically suppresses tif-mediated SOS induction implies a role of the replication apparatus in this process, as has been suggested for ultraviolet induction. The induction of lambda is known to proceed via repressor cleavage, presumably promoted by an activated (protease) form of the recA protein. Since lambda induction is normal after ultraviolet irradiation of the tif-1 dnaB252(lambda) strain, tif-mediated induction in this strain may be blocked in a tif-specific step leading to activation of the recA (tif) protein. It is possible that the recA (tif) mutant protein may be directly involved in the replication complex in processes leading to this activation.     

The dnaB-dnaC replication protein complex of Escherichia coli. II. Role of the complex in mobilizing dnaB functions

The dnaC protein of Escherichia coli, by forming a complex with the dnaB protein, facilitates the interactions with single-stranded DNA that enable dnaB to perform its ATPase, helicase, and priming functions. Within the dnaB-dnaC complex, dnaB appears to be inactive but becomes active upon the ATP-dependent release of dnaC from the complex. With adenosine 5'-(gamma-thio)triphosphate substituted for ATP, the dnaB-dnaC complex does not direct dnaB to its targeted actions. Excess dnaC inhibits dna beta actions and augments the ATP gamma S effects. In the dnaA protein-driven initiation of duplex chromosome replication, dnaB is introduced for its essential helicase role via the dnaB-dnaC complex. Similarly, when the dnaA protein interacts nonspecifically with single-stranded DNA, the dnaB-dnaC complex is essential to introduce dnaB for its role in primer formation by primase.     

The Mycobacterium avium-intracellulare complex dnaB locus and protein intein splicing

Intein is a protein sequence mebedded in-frame within a precursor protein and is posttranslationally excised by a self-catalytic protein splicing process. Protein splicing is believed to follow a pathway requiring Cys, Ser, or Thr residues at the intein N-terminus and substitutions other than Cys, Ser, or Thr residues prevent splicing. We show that the dnaB locus in some strains of M. avium-intracellulare complex (MAC) contains intein and that the intein N-terminal amino acid is Ala [Ala-type]. We demonstrate that the M. avium DnaB precursor protein undergoes posttranslational proteolytic processing producing proteins corresponding to the sizes of the DnaB and intein. Further, by Western analysis we detect a protein corresponding to the size of the spliced DnaB protein in MAC cell extracts. Together, these results indicate that the Ala-type MAC DnaB inteins can splice and provide another example that points to an interesting alternative splicing mechanism (Southworth, M. W., Benner, J., and Perler, F. B., EMBO J. 19, 5019-5026, 2000).     

Stabilization by ATP and ADP of Escherichia coli dnaB protein activity

The effect of adenine ribonucleotides on the stability of Escherichia coli dnaB protein in cellular crude extracts was studied. Stabilization of dnaB protein by ATP or ADP, but not by AMP, was manifested in that (i) the activity and yield of wild type dnaB protein is enhanced in the presence of ATP, (ii) the dnaB protein of E. coli dnaB mutants, such as groPB and dnaB252/ColE1::dnaC+, which is inactive in a dnaB complementation assay, can be isolated in active form in the presence of ATP or aDP, (iii) ATP or ADP protect the dnaB protein of an E. coli dnaBts mutant from inactivation at 37 degrees C, and (iv) inactive groPB and dnaBts protein can be reactivated partially by ATP. Thus, the stabilizing effect of ATP and ADP can be exploited for the isolated of otherwise inactive or labile mutant dnaB proteins.     

The Escherichia coli dnaB replication protein is a DNA helicase

Genetic and biochemical analyses indicate that the Escherichia coli dnaB replication protein functions in the propagation of replication forks in the bacterial chromosome. We have found that the dnaB protein is a DNA helicase that is capable of unwinding extensive stretches of double-stranded DNA. We constructed a partially duplex DNA substrate, containing two preformed forks of single-stranded DNA, which was used to characterize this helicase activity. The dnaB helicase depends on the presence of a hydrolyzable ribonucleoside triphosphate, is maximally stimulated by a combination of E. coli single-stranded DNA-binding protein and E. coli primase, is inhibited by antibody directed against dnaB protein, and is inhibited by prior coating of the single-stranded regions of the helicase substrate with the E. coli single-stranded DNA-binding protein. It was determined that the dnaB protein moves 5' to 3' along single-stranded DNA, apparently in a processive fashion. To invade the duplex portion of the helicase substrate, the dnaB protein requires a 3'-terminal extension of single-stranded DNA in the strand to which it is not bound. Under optimal conditions at 30 degrees C, greater than 1 kilobase pair of duplex DNA can be unwound within 30 s. Based on these findings and other available data, we propose that the dnaB protein is the primary replicative helicase of E. coli and that it actively and processively migrates along the lagging strand template, serving both to unwind the DNA duplex in advance of the leading strand and to potentiate synthesis by the bacterial primase of RNA primers for the nascent (Okazaki) fragments of the lagging strand.     

Structure of Escherichia coli dnaC. Identification of a cysteine residue possibly involved in association with dnaB protein

The nucleotide sequence of the Escherichia coli dnaC gene and the primary structure of the dnaC protein were determined. The NH2-terminal amino acid sequence of the dnaC protein matched that predicted from the nucleotide sequence of the 735-base pair coding region. The dnaC gene lacks characteristic promoter structures; neither the "Pribnow box" nor the "-35 sequence" was detected within 222 base pairs upstream from the initiator ATG codon. There is, however, a typical Shine-Dalgarno sequence 7-10 base pairs before the ATG codon. An upstream open reading frame, separated by just 2 base pairs from the coding region of dnaC, encodes the COOH-terminal half of the dnaT product (protein i; Masai, H., Bond, M. W., and Arai, K. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1256-1260). The dnaC protein contains 245 amino acids with a calculated molecular weight of 27,894 consistent with the observed value (29,000). Similar to dnaG and dnaT, dnaC uses several minor codons; the significance of these minor codons to the low level expression of the protein product in E. coli cells remains to be determined. The in vitro site-directed mutagenesis method was employed to determine the functional region involved in interaction with dnaB protein. The first cysteine residue located in the NH2-terminal region of the dnaC protein (Cys69) was shown to be important for this activity. Overall sequence homology between dnaC protein and lambda P protein, functionally analogous to the dnaC protein in the lambda phage DNA replication, is not extensive. There are, however, several short stretches of homologous regions including the NH2-terminal eight amino acids and the Cys78 region of dnaC protein.