The E. coli replication fork synthesizes DNA at the rate of nearly 1000 nt/s. We show here that an interaction between the tau subunit of the replicative polymerase (the DNA polymerase III holoenzyme) and the replication fork DNA helicase (DnaB) is required to mediate this high rate of replication fork movement. In the absence of this interaction, the polymerase follows behind the helicase at a rate equal to the slow (approximately 35 nt/s) unwinding rate of the helicase alone, whereas upon establishing a tau-DnaB contact, DnaB becomes a more effective helicase, increasing its translocation rate by more than 10-fold. This finding establishes the existence of both a physical and communications link between the two major replication machines in the replisome: the DNA polymerase and the primosome.

A study of the properties of the complex containing 5-fluoro-2 '-deoxyuridylate (FdUMP), 5,10-methylenetetrahydrofolate, and thymidylate synthetase is described. In the presence of the cofactor, isolable complexes contain two tightly bound molecules of FdUMP per enzyme molecule of 70,000 daltons. A number of folate analogs also stimulate binding of FdUMP, albeit to a lesser degree than the cofactor. Kinetic data indicate the rate constant for association of FdUMP with the enzyme-methylenetetrahydrofolate complex to be 2 X 107 m_1 min""* 1 at 24°. The unimolecular dissociation rates of FdUMP from the complex are highly temperature dependent and show * = 21.5 kcal/mol, /7= = 28.4 kcal/mol, and 5* = 0.023 eu; there is no indication that homotropic interactions, if existent, are manifested in the rate of dissociation of FdUMP. From kinetic data, an assocation constant for the interaction of FdUMP with the enzyme-cofactor complex is calculated to be ca. 2 X 1010 nt1 at 24°. Within the enzyme-cofactor-FdUMP complex, a t From the

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The T and y subunits of DNA polymerase Iml holoenzyme are both products of the dnaX gene. Since X and y are required as stoichiometric components of the replicative complex, a mechanism must exist for the cell to coordinate their synthesis and ensure that both subunits are present in an adequate quantity and ratio for assembly. We have proposed that y is produced by a translational frameshift event. In this report, we describe the use of dnaX-lacZ fusions in all three reading frames to demonstrate that v, the shorter product of dnaX, is generated by ribosomal frameshifting to the -1 reading frame of the mRNA within an oligo(A) sequence that is followed by a sequence predicted to form a stable secondary structure. Immediately after frameshifting a stop codon is encountered, leading to translational termination. Mutagenesis of the oligo(A) sequence abolishes frameshifting, and partial disruption of the predicted distal secondary structure severely impairs the efficiency. Comparison of the expression of lacZ fused to dnaX distal to the site of frameshifting in the -1 and o reading frames indicates that the efficiency of frameshifting is -40%.DNA polymerase III holoenzyme is the major replicative enzyme in Escherichia coli. It is a complex enzyme consisting of at least seven distinct subunits, each required for full reconstitution of holoenzyme activity in vitro (for a review, see ref. 1). The structural genes for five of the subunits have been identified and their nucleic acid sequences have been determined. The structural gene for the a subunit is dnaE (2, 3); dnaN is the gene for the /8 subunit (4, 5); E is encoded by dnaQ (mutD) (6-10); and the r and y subunits are both encoded by dnaX (11-15).Based on complementation, dnaX was originally defined as two distinct genes, dnaX and dnaZ (12). The dnaZ gene product was identified to be y by in vitro complementation of inactive extracts of temperature-sensitive dnaZ mutants (11,13) and by the overproduction of y from an expression vector containing dnaZ (13, 16). The dnaX gene product was first identified to be rbased on the comigration of rand the labeled dnaX gene product from "maxicells" during electrophoresis (14, 15). This conclusion was confirmed by immunoprecipitation of the dnaX gene product with a monoclonal antibody directed against r (17).A 2.2-kilobase restriction fragment can complement both dnaZ and dnaX mutations and direct the synthesis of both the y and X subunits (14,15). In a single open reading frame, 2.2 kilobases can encode no more than 80,000 daltons of protein.The molecular weights of r and y are 71,000 and 52,000, respectively; the sum of these is clearly in excess of the coding capacity of the region. Deletion analysis and partial tryptic digests indicated that r and y share a common amino terminus (14,15 (18,19). Since dnaZ is contained within dnaX, it has been proposed that the use of dnaZ be discontinued and that the gene should be designated dnaX (20). A similar situation may exist in Salmonella typhimurium, where the dnaZ and dna...