Grażyna Konopa

Identification and characterization of single- stranded-DNA-binding proteins from Thermus thermophilus and Thermus aquaticus - new arrangement of binding domains

Single-stranded-DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination and repair in bacteria, archaea and eukarya. This paper reports the identification and characterization of the SSB-like proteins of the thermophilic bacteria Thermus thermophilus and Thermus aquaticus. These proteins (TthSSB and TaqSSB), in contrast to their known counterparts from mesophilic bacteria, archaea and eukarya, are homodimers, and each monomer contains two ssDNA-binding domains with a conserved OB (oligonucleotide/oligosaccharide-binding) fold, as deduced from the sequence analysis. The N-terminal domain is located in the region from amino acid 1 to 123 and the C-terminal domain is located between amino acids 124 and 264 or 266 in TthSSB and TaqSSB, respectively. Purified TthSSB or TaqSSB binds only to ssDNA and with high affinity. The binding site size for TaqSSB and TthSSB protein corresponds to 30-35 nucleotides. It is concluded that the SSBs of thermophilic and mesophilic bacteria, archaea and eukarya share a common core ssDNA-binding domain. This ssDNA-binding domain was presumably present in the common ancestor to all three major branches of life.

Sulforaphane, a cruciferous vegetable-derived isothiocyanate, inhibits protein synthesis in human prostate cancer cells

Sulforaphane (SFN) is a compound derived from cruciferous plants. Its anticancer properties have been demonstrated both, in cancer cell lines as well as tumors in animal models. It has been shown that SFN inhibits cell proliferation, induces apoptosis, autophagy, and sensitizes cancer cells to therapies. As induction of catabolic processes is often related to perturbation in protein synthesis we aimed to investigate the impact of SFN on this process in PC-3 human prostate cancer cells. In the present study we show that SFN inhibits protein synthesis in PC-3 cells in a dose- and time-dependent manner which is accompanied by a decreased phosphorylation of mTOR substrates. Translation inhibition is independent of mitochondria-derived ROS as it is observed in PC-3 derivatives devoid of functional mitochondrial respiratory chain (Rho0 cells). Although SFN affects mitochondria and slightly decreases glycolysis, the ATP level is maintained on the level characteristic for control cells. Inhibition of protein synthesis might be a protective response of prostate cancer cells to save energy. However, translation inhibition contributes to the death of PC-3 cells due to decreased level of a short-lived protein, survivin. Overexpression of this anti-apoptotic factor protects PC-3 cells against SFN cytotoxicity. Protein synthesis inhibition by SFN is not restricted to prostate cancer cells as we observed similar effect in SKBR-3 breast cancer cell line.

Impaired chromosome partitioning and synchronization of DNA replication initiation in an insertional mutant in the Vibrio harveyi cgtA gene coding for a common GTP-binding protein.

The Vibrio harveyi cgtA gene product belongs to a subfamily of small GTP-binding proteins, called Obg-like proteins. Members of this subfamily are present in diverse organisms ranging from bacteria to humans. On the other hand, the functions of these proteins in the regulation of cellular processes are largely unknown. Genes coding for these proteins are essential in almost all bacteria investigated thus far. However, a viable V. harveyi insertional mutant in the cgtA gene was described recently. Therefore, this mutant gives a unique opportunity to study functions of a member of the subfamily of Obg-like proteins. Here we demonstrate that the mutant cells often form long filaments with expanded, non-partitioned or rarely partitioned chromosomes. Such a phenotype suggests impairment of the mechanism of chromosome partition. Flow cytometric studies revealed that synchronization of chromosome replication initiation is also significantly disturbed in the cgtA mutant. Moreover, in contrast to wild-type V. harveyi, inhibition of chromosome replication and/or of cell division in the mutant bacteria caused significant increase in the number of large cells, suggesting that the cgtA gene product may be involved in the coupling of cell growth to chromosome replication and cell division. These results indicate that CgtA, an Obg-like GTP-binding protein, plays an important role in the regulation of chromosomal functions.

DnaA Box Sequences as the Site for Helicase Delivery during Plasmid RK2 Replication Initiation in Escherichia coli

DnaA box sequences are a common motif present within the replication origin region of a diverse group of bacteria and prokaryotic extrachromosomal genetic elements. Although the origin opening caused by binding of the host DnaA protein has been shown to be critical for the loading of the DnaB helicase, to date there has been no direct evidence presented for the formation of the DnaB complex at the DnaA box site. For these studies, we used the replication origin of plasmid RK2 (oriV), containing a cluster of four DnaA boxes that bind DnaA proteins isolated from different bacterial species (Caspi, R., Helinski, D. R., Pacek, M., and Konieczny, I. (2000) J. Biol. Chem. 275, 18454–18461). Size exclusion chromatography, surface plasmon resonance, and electron microscopy experiments demonstrated that the DnaB helicase is delivered to the DnaA box region, which is localized ∼200 base pairs upstream from the region of origin opening and a potential site for helicase entry. The DnaABC complex was formed on both double-stranded superhelical and linear RK2 templates. A strict DnaA box sequence requirement for stable formation of that nucleoprotein structure was confirmed. In addition, our experiments provide evidence for interaction between the plasmid initiation protein TrfA and the DnaABC prepriming complex, formed at DnaA box region. This interaction is facilitated via direct contact between TrfA and DnaB proteins.

A Vibrio harveyi insertional mutant in the cgtA (obg, yhbZ) gene, whose homologues are present in diverse organisms ranging from bacteria to humans and are essential genes in many bacterial species

The cgtA gene product is a member of the subfamily of small GTP-binding proteins that have been identified in diverse organisms ranging from bacteria to humans. In bacteria that sporulate or display another special developmental programme, this gene (referred to as cgtA, obg or yhbZ) appears to be involved in the regulation of these processes. However, this gene has also been found to be essential in all bacterial species investigated to date, although its role in bacteria that do not sporulate and do not undergo a specific development remains unknown. Here the authors characterize a Vibrio harveyi mutant bearing a transposon insertion into the cgtA gene. This mutant reveals a multiple phenotype: it grows more slowly than the wild-type strain in a rich medium; its growth is completely inhibited in minimal media; its survival in 3% NaCl is dramatically reduced; it is very sensitive to UV irradiation; it is more susceptible to mutation upon treatment with different mutagens; its luminescence is decreased; its quorum-sensing regulation is less effective than in the wild-type strain; and the elongated shape of the mutant cells may suggest problems with the regulation of cell division and/or DNA replication. These defects in diverse cellular processes found in the insertional cgtA mutant of V. harveyi indicate that in a bacterium that does not sporulate and does not display other special development programmes, the CgtA protein is involved in the regulation of many crucial biochemical reactions, possibly at the stage of signal transduction.

SeqA, the Escherichia coli origin sequestration protein, is also a specific transcription factor

The SeqA protein is a negative regulator of initiation of DNA replication in the Escherichia coli chromosome. Here, we demonstrate that SeqA stimulates transcription from the bacteriophage λpR promoter both in vivo and in vitro. The activity of the λpL promoter was found not to be affected by this protein. SeqA‐mediated stimulation of pR was dependent on the state of template methylation: transcription was activated on fully methylated and hemimethylated templates but not on an unmethylated template. Using electrophoretic mobility shift assay and electron microscopy, we demonstrated that SeqA interacts specifically with a pR promoter region located on both fully methylated and hemimethylated DNA molecules, but not on unmethylated DNA. The activity of SeqA was found to affect the initiation of λ plasmid replication positively in vivo, probably via pR‐dependent expression of λ replication genes and transcriptional activation of ori λ. We conclude that, apart from its function in the control of DNA replication, SeqA is also a specific transcription factor.

INTERACTION OF THE ESCHERICHIA COLI DNAA PROTEIN WITH BACTERIOPHAGE LAMBDA DNA

Abstract Interaction of the Escherichia coli DnaA (replication initiator) protein with restriction fragments of phage λ DNA demonstrated differential binding of DnaA along the whole λ DNA. Interaction of DnaA with the λ replication region (from the promoter pR to the origin of replication, oriλ) demonstrated a strong binding of DnaA to the region around the po promoter where synthesis of a short antisense oop RNA is initiated. The four sequences protected by DnaA (two 9mers and two 5mers) are not related even to a relaxed DnaA box. The pattern of protection of these four sequences and the location of three DNase I hypersensitive sites in the λ DNA r strand, together with results of mobility shift assays and electron microscopy studies, may indicate an interaction involving DnaA monomers bound to different DNA positions on one side of the helix and the formation of higher-order nucleoprotein structures. Therefore, it is tempting to suggest that DnaA, in addition to its activity in regulation of replication and transcription, could be considered as a factor which structures certain chromosomal regions.

Regulation of the switch from early to late bacteriophage lambda DNA replication.

There are two modes of bacteriophage lambda DNA replication following infection of its host, Escherichia coli. Early after infection, replication occurs according to the theta (theta or circle-to-circle) mode, and is later switched to the sigma (sigma or rolling-circle) mode. It is not known how this switch, occurring at a specific time in the infection cycle, is regulated. Here it is demonstrated that in wild-type cells the replication starting from orilambda proceeds both bidirectionally and unidirectionally, whereas in bacteria devoid of a functional DnaA protein, replication from orilambda is predominantly unidirectional. The regulation of directionality of replication from orilambda is mediated by positive control of lambda p(R) promoter activity by DnaA, since the mode of replication of an artificial lambda replicon bearing the p(tet) promoter instead of p(R) was found to be independent of DnaA function. These findings and results of density-shift experiments suggest that in dnaA mutants infected with lambda, phage DNA replication proceeds predominantly according to the unidirectional theta mechanism and is switched early after infection to the sigma mode. It is proposed that in wild-type E. coli cells infected with lambda, phage DNA replication proceeds according to a bidirectional theta mechanism early after infection due to efficient transcriptional activation of orilambda, stimulated by the host DnaA protein. After a few rounds of this type of replication, the resulting increased copy number of lambda genomic DNA may cause a depletion of free DnaA protein because of its interaction with the multiple DnaA-binding sites in lambda DNA. It is proposed that this may lead to inefficient transcriptional activation of orilambda resulting in unidirectional theta replication followed by sigma type replication.

Changes in hair morphology of mucopolysaccharidosis I patients treated with recombinant human α-L-iduronidase (laronidase, Aldurazyme)

Mucopolysaccharidoses (MPS) are heritable, metabolic diseases caused by accumulation of mucopolysaccharides (glycosaminoglycans, GAGs) in lysosomes. This accumulation is due to a deficiency in one of several specific enzymes involved in the degradation of GAGs. MPS type I (MPS I) is caused by low or undetectable activity of α‐L‐iduronidase, an enzyme involved in removing the terminal iduronic acid residues from heparan and dermatan sulfate. Recently, an enzyme replacement therapy (ERT) for MPS I, based on administration of recombinant human α‐L‐iduronidase (laronidase, Aldurazyme), became available. The assessment of efficacy of ERT is especially important because MPS I is a highly variable and very rare disease, and the clinical trials involved relatively low number of patients. Among various significant clinical improvements during ERT, remarkable changes in hair morphology were noted. Detailed studies of hair samples from one patient, who did not have a hair cut from the beginning of ERT to the end of this study, and supported by results obtained for two other patients, revealed hair shaft structural abnormalities in MPS I hair. These hair abnormalities disappeared upon treatment with Aldurazyme. Although hair morphology is of limited clinical importance, the data suggest that changes in this parameter could be a useful, additional tool for a rapid, non‐invasive, preliminary assessment of ERT efficacy.

SeqA‐mediated stimulation of a promoter activity by facilitating functions of a transcription activator

It was demonstrated recently that the SeqA protein, a main negative regulator of Escherichia coli chromosome replication initiation, is also a specific transcription factor. SeqA specifically activates the bacteriophage λpR promoter while revealing no significant effect on the activity of another λ promoter, pL. Here, we demonstrate that lysogenization by bacteriophage λ is impaired in E. coli seqA mutants. Genetic analysis demonstrated that CII‐mediated activation of the phage pI and paQ promoters, which are required for efficient lysogenization, is less efficient in the absence of seqA function. This was confirmed in in vitro transcription assays. Interestingly, SeqA stimulated CII‐dependent transcription from pI and paQ when it was added to the reaction mixture before CII, although having little effect if added after a preincubation of CII with the DNA template. This SeqA‐mediated stimulation was absolutely dependent on DNA methylation, as no effects of this protein were observed when using unmethylated DNA templates. Also, no effects of SeqA on transcription from pI and paQ were observed in the absence of CII. Binding of SeqA to templates containing the tested promoters occurs at GATC sequences located downstream of promoters, as revealed by electron microscopic studies. In contrast to pI and paQ, the activity of the third CII‐dependent promoter, pE, devoid of neighbouring downstream GATC sequences, was not affected by SeqA both in vivo and in vitro. We conclude that SeqA stimulates transcription from pI and paQ promoters in co‐operation with CII by facilitating functions of this transcription activator, most probably by allowing more efficient binding of CII to the promoter region.