Phenotypes of Mutant Cells Indicate that the Escherichia coli Clamp Loader Has a Role in the Restart of Stalled Replication Forks.

The mutation was discovered in connection with a screen for multicopy suppressors of the temperature-sensitive topoisomerase IV mutation The gene for the clamp loader subunits τ and γ, , but not the mutant , was found to suppress (Ts) when overexpressed. Purified mutant protein was found to be functional , and few phenotypes were found apart from problems with partitioning of DNA in rich medium. We show here that a large number of the replication forks that initiate at never reach the terminus in mutant cells.

SeqA structures behind Escherichia coli replication forks affect replication elongation and restart mechanisms.

The SeqA protein binds hemi-methylated GATC sites and forms structures that sequester newly replicated origins and trail the replication forks. Cells that lack SeqA display signs of replication fork disintegration. The broken forks could arise because of over-initiation (the launching of too many forks) or lack of dynamic SeqA structures trailing the forks.

The DnaA Protein Is Not the Limiting Factor for Initiation of Replication in Escherichia coli.

The bacterial replication cycle is driven by the DnaA protein which cycles between the active ATP-bound form and the inactive ADP-bound form. It has been suggested that DnaA also is the main controller of initiation frequency. Initiation is thought to occur when enough ATP-DnaA has accumulated.

The Escherichia coli datA site promotes proper regulation of cell division.

In Escherichia coli inhibition of replication leads to a block of cell division. This checkpoint mechanism ensures that no cell divides without having two complete copies of the genome to pass on to the two daughter cells. The chromosomal datA site is a 1 kb region that contains binding sites for the DnaA replication initiator protein, and which contributes to the inactivation of DnaA.

The Fis protein has a stimulating role in initiation of replication in Escherichia coli in vivo.

The Fis protein is a nucleoid associated protein that has previously been reported to act negatively in initiation of replication in Escherichia coli. In this work we have examined the influence of this protein on the initiation of replication under different growth conditions using flow cytometry. The Fis protein was found to be increasingly important with increasing growth rate.

Depletion of acidic phospholipids influences chromosomal replication in Escherichia coli.

In Escherichia coli, coordinated activation and deactivation of DnaA allows for proper timing of the initiation of chromosomal synthesis at the origin of replication (oriC) and assures initiation occurs once per cell cycle. In vitro, acidic phospholipids reactivate DnaA, and in vivo depletion of acidic phospholipids, results in growth arrest. Growth can be restored by the expression of a mutant form of DnaA, DnaA(L366K), or by oriC-independent DNA synthesis, suggesting acidic phospholipids are required for DnaA- and oriC-dependent replication.

An easy-to-use simulation program demonstrates variations in bacterial cell cycle parameters depending on medium and temperature.

Many studies are performed on chromosome replication and segregation in Escherichia coli and other bacteria capable of complex replication with C phases spanning several generations. For such investigations an understanding of the replication patterns, including copy numbers of origins and replication forks, is crucial for correct interpretation of the results.Flow cytometry is an important tool for generation of experimental DNA distributions of cell populations.

The G157C mutation in the Escherichia coli sliding clamp specifically affects initiation of replication.

Escherichia coli cells with a point mutation in the dnaN gene causing the amino acid change Gly157 to Cys, were found to underinitiate replication and grow with a reduced origin and DNA concentration. The mutant β clamp also caused excessive conversion of ATP-DnaA to ADP-DnaA. The DnaA protein was, however, not the element limiting initiation of replication.

DnaA protein interacts with RNA polymerase and partially protects it from the effect of rifampicin.

The Escherichia coli DnaA protein forms an oligomer at the origin and initiates chromosome replication with the aid of architectural elements and transcription by RNA polymerase. Rifampicin inhibits initiation of transcription by RNA polymerase and thus also initiation of replication. Here, we report that wild-type cells undergo rifampicin-resistant initiation of replication during slow growth in acetate medium.