Open Questions about the Roles of DnaA, Related Proteins, and Hyperstructure Dynamics in the Cell Cycle.

The DnaA protein has long been considered to play the key role in the initiation of chromosome replication in modern bacteria. Many questions about this role, however, remain unanswered. Here, we raise these questions within a framework based on the dynamics of hyperstructures, alias large assemblies of molecules and macromolecules that perform a function.

Research on DnaA in the early days.

The Escherichia coli chromosome is a circular double helix. DNA polymerase, therefore, cannot use it directly as a template for polymerization until it has first been unwound. The DnaA protein opens the chromosome at a unique and specific site (oriC), which allows the polymerase to begin DNA replication.

Trashing of single-stranded DNA generated during processing of arrested replication fork in E. coli.

We analyzed formation of single-stranded DNA (ssDNA) related to SOS induction in nalidixilate (Nal)-treated Escherichia coli, using immunofluorescence microscopy accompanied by computer analysis. We found enhancement of both ssDNA concentrations and cells having ssDNA foci that often localized around cellpoles. Analyzing several mutants deficient in DNA repair or replication, we found, after Nal treatment, that recN, recA, uvrD and dnaB failed to increase ssDNA concentration and that recG and particularly ruvA only partially enhanced it.

DNA replication defects in a mutant deficient in the thioredoxin homolog YbbN.

Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, that displays both redox and chaperone properties. Since three out of the six proteins of the YbbN interactome (Butland et al., 2005) are components of DNA polymerase 3 holoenzyme (i.

Generation of DNA-free Escherichia coli cells by 2-aminopurine requires mismatch repair and nonmethylated DNA.

Undirected mismatch repair initiated by the incorporation of the base analog 2-aminopurine kills DNA-methylation-deficient Escherichia coli dam cells by DNA double-strand breakage. Subsequently, the chromosomal DNA is totally degraded, resulting in DNA-free cells.

Localization of replication forks in wild-type and mukB mutant cells of Escherichia coli.

To examine the subcellular localization of the replication machinery in Escherichia coli, we have developed an immunofluorescence method that allows us to determine the subcellular location of newly synthesized DNA pulse-labeled with 5-bromo-2'-deoxyuridine (BrdU). Using this technique, we have analyzed growing cells. In wild-type cells that showed a single BrdU fluorescence signal, the focus was located in the middle of the cell; in cells with two signals, the foci were localized at positions equivalent to 1/4 and 3/4 of the cell length.

Bacterial sex: playing voyeurs 50 years later.

The concept of chromosomes with a ring structure was born during the early studies of bacterial sexuality, and the discovery of fertility factors- episomes or plasmids-provided much later the key tools for gene cloning and biotechnology. But the plasmid-mediated transfer of antibiotic and other resistances, as well as pathogenicity, has served bacteria well in their own adaptive evolution.

Isolation of a new hemimethylated DNA binding protein which regulates dnaA gene expression.

In this report, we show that yccV, a gene of unknown function, encodes a protein having an affinity for a hemimethylated oriC DNA and that the protein negatively controls dnaA gene expression in vivo.

Specificity of elongation factor EF-TU for hydrophobic peptides.

The elongation factor EF-Tu carries aminoacyl-tRNAs to the A-site of the ribosome during the elongation process of protein biosynthesis. We, and others, have recently reported that the Escherichia coli EF-Tu interacts with unfolded and denatured proteins and behaves like a chaperone in protein folding and protection against protein thermal denaturation. In this study, we have identified EF-Tu binding sites in protein substrates by screening cellulose-bound peptides scanning the sequences of several proteins.