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In all domains of life, DNA synthesis occurs bidirectionally from replication origins. Despite variable rates of replication fork progression, fork convergence often occurs at specific sites (1). Escherichia coli sets a 'replication fork trap' that allows the first arriving fork to enter but not to leave the terminus region (2-5). The trap is set by oppositely oriented Tus-bound Ter sites that block forks on approach from only one direction (3-7). However, the efficiency of fork blockage by Tus-Ter does not exceed 50% in vivo despite its apparent ability to almost permanently arrest replication forks in vitro (8, 9). Here we use data from single-molecule DNA replication assays and structural studies to show that both polarity and fork-arrest efficiency are determined by a competition between rates of Tus displacement and rearrangement of Tus-Ter interactions that leads to blockage of slower moving replisomes by two distinct mechanisms. To our knowledge this is the first example where intrinsic differences in rates of individual replisomes have different biological outcomes., In the circular E. coli chromosome, two replication forks move from the replication origin to converge opposite in a region that contains ten 23-base-pair Ter (termination) sites and the dif [...]

The structure of the 30 kDa complex between a lanthanide [Ln.sup.3}-labeled protein, the N-terminal domain of the subunit epsilon bound to the subunit theta of the Escherichia coli DNA polymerase III, and a small ligand molecule is reported. The bounding mode of thymidine is found to be very similar to that of thymidine monophosphate present in the crystal structure.

The rates of dissociation of Tus from forked TerB oligonucleotides, which would be produced by the replicative DnaB helicase at both the fork-blocking and permissive ends of the Ter site, are measured to study the basis of polarity. The analysis has explained the mechanism of replication fork arrest at Tus bound Ter sites in the final stage of chromosomal DNA replication and has also revealed the stable interaction between a monomeric DNA binding protein and a forked DNA structure.

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits, the [alpha], [epsilon], and [theta] subunits. The [theta] subunit is the smallest and least understood subunit. The three-dimensional structure of [theta] in a complex with the unlabeled N-terminal domain of the e subunit, [epsilon] 186, was determined by multidimensional nuclear magnetic resonance spectroscopy. The structure was refined using pseudocontact shifts that resulted from inserting a lanthanide ion ([Dy.sup.3+], [Er.sup.3+], or [Ho.sup.3+]) at the active site of [epsilon] 186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of [theta] in a methanolwater buffer and of the bacteriophage P1 homolog, HOT, in aqueous buffer. Conserved nuclear Overhauser enhancement (NOE) patterns obtained for free and complexed [theta] show that most of the structure changes little upon complex formation. Discrepancies with respect to a previously published structure of free [theta] (Keniry et al., Protein Sci. 9:721-733, 2000) were attributed to errors in the latter structure. The present structure satisfies the pseudocontact shifts better than either the structure of [theta] in methanol-water buffer or the structure of HOT. satisfies these shifts. The epitope of [epsilon]186 on [theta] was mapped by NOE difference spectroscopy and was found to involve helix 1 and the C-terminal part of helix 3. The pseudocontact shifts indicated that the helices of [theta] are located about 15 [Angstrom] or farther from the lanthanide ion in the active site of [epsilon] 186, in agreement with the extensive biochemical data for the [theta]-[epsilon] system.

Author(s): Christopher M. Elvin (corresponding author) [1]; Andrew G. Carr [1]; Mickey G. Huson [2]; Jane M. Maxwell [2]; Roger D. Pearson [1]; Tony Vuocolo [1]; Nancy E. Liyou [1]; [...]

A historical overview of the development of the model for the process of replication in Escherichia coli is presented and the hypotheses concerning the mechanism by which interaction of the replication terminator protein (Tus) at Ter sites leads to polar arrest of advancing replications forks is examined. Results reveal that while it is possible to explain polar fork arrest by a mechanism involving only the Tus-Ter interaction, there are also strong indications of a role for specific Tus-DnaB interactions.

Results indicate that the epsilon subunit of the DNA polymerase, epsilon186 and the theta complex is less active on nitrophenyl ester-thymidine 5'-monophosphate (TMP) hydrolysis than epsilon186 alone. Data show that hydrolysis of the nitrophenyl ester requires metal ion cofactors and is inhibited by TMP.

A model for the interaction of the E. coli Tus replication terminator protein with the TerB DNA sequence is described. Binding involves structural changes in the protein, as shown by surface plasmon resonance.