Biomedical subjects

Anna Zawilak

Publications and source records attributed to Anna Zawilak.

3 recordsLinked to original sources

PubMed2004

Mycobacterium tuberculosis DnaA initiator protein: purification and DNA-binding requirements.

The Mycobacterium tuberculosis oriC (the origin of chromosomal replication) region contains 13 non-perfect DnaA boxes. The M. tuberculosis initiator protein, DnaA, was overexpressed in Escherichia coli as a soluble His-tagged fusion protein. The purified protein His6MtDnaA was investigated for its binding properties to DnaA boxes from the oriC region. Gel retardation demonstrated that the DnaA from M. tuberculosis requires two DnaA boxes for efficient binding. Electron microscopy as well as DNase I footprinting showed that the His6MtDnaA protein binds to four specific regions, which correspond to the location of 11 out of 13 previously identified DnaA boxes within the M. tuberculosis oriC. Probably, in M. tuberculosis, DnaA molecules by co-operative binding of numerous 'non-perfect' DnaA boxes assemble along the oriC region and subsequently form a massive nucleoprotein complex.

Bacterial Proteins↗

PubMed2004

Where does bacterial replication start? Rules for predicting the oriC region.

Three methods, based on DNA asymmetry, the distribution of DnaA boxes and dnaA gene location, were applied to identify the putative replication origins in 120 chromosomes. The chromosomes were classified according to the agreement of these methods and the applicability of these methods was evaluated. DNA asymmetry is the most universal method of putative oriC identification in bacterial chromosomes, but it should be applied together with other methods to achieve better prediction. The three methods identify the same region as a putative origin in all Bacilli and Clostridia, many Actinobacteria and gamma Proteobacteria. The organization of clusters of DnaA boxes was analysed in detail. For 76 chromosomes, a DNA fragment containing multiple DnaA boxes was identified as a putative origin region. Most bacterial chromosomes exhibit an overrepresentation of DnaA boxes; many of them contain at least two clusters of DnaA boxes in the vicinity of the oriC region. The additional clusters of DnaA boxes are probably involved in controlling replication initiation. Surprisingly, the characteristic features of the initiation of replication, i.e. a cluster of DnaA boxes, a dnaA gene and a switch in asymmetry, were not found in some of the analysed chromosomes, particularly those of obligatory intracellular parasites or endosymbionts. This is presumably connected with many mechanisms disturbing DNA asymmetry, translocation or disappearance of the dnaA gene and decay of the Escherichia coli perfect DnaA box pattern.

Bacteria↗

PubMed2003

DNA binding specificity of the replication initiator protein, DnaA from Helicobacter pylori.

The key protein in the initiation of Helicobacter pylori chromosome replication, DnaA, has been characterized. The amount of the DnaA protein was estimated to be approximately 3000 molecules per single cell; a large part of the protein was found in the inner membrane. The H.pylori DnaA protein has been analysed using in vitro (gel retardation assay and surface plasmon resonance (SPR)) as well as in silico (comparative computer modeling) studies. DnaA binds a single DnaA box as a monomer, while binding to the fragment containing several DnaA box motifs, the oriC region, leads to the formation of high molecular mass nucleoprotein complexes. In comparison with the Escherichia coli DnaA, the H.pylori DnaA protein exhibits lower DNA-binding specificity; however, it prefers oriC over non-box DNA fragments. As determined by gel retardation techniques, the H.pylori DnaA binds with a moderate level of affinity to its origin of replication (4nM). Comparative computer modelling showed that there are nine residues within the binding domain which are possible determinants of the reduced H.pylori DnaA specificity. Of these, the most interesting is probably the triad PTL; all three residues show significant divergence from the consensus, and Thr398 is the most divergent residue of all.

Bacterial Proteins↗