Sylwia Barańska

Genistein-mediated inhibition of glycosaminoglycan synthesis as a basis for gene expression-targeted isoflavone therapy for mucopolysaccharidoses

Mucopolysaccharidoses (MPS) are inherited, severe, progressive, metabolic disorders caused by deficiencies in different enzymes involved in degradation of glycosaminoglycans (GAGs). Although enzyme replacement therapy (ERT) has recently been available for MPS type I, and clinical trials have been performed in ERT for MPS II and MPS VI, there is little chance that this kind of treatment may be effective for neurodegenerative forms of MPS (due to inefficient delivery of enzymes to central nervous system through the blood–brain barrier), hence currently there is no effective therapy available for them. Therefore, we aim to develop an alternative therapy for these diseases. We found that genistein (4′,5,7-trihydroxyisoflavone or 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) inhibits synthesis of GAGs considerably in cultures of fibroblasts of MPS patients (types I, II, IIIA and IIIB were tested). Prolonged cultivation of these cells in the presence of genistein resulted in reduction of GAG accumulation and normalization of cells as estimated by biochemical tests and electron microscopic analysis, respectively. As genistein inhibits kinase activity of epidermal growth factor receptor, which is required for full expression of genes coding for enzymes involved in GAG production, we propose to consider a substrate reduction therapy for MPS, which is referred to as ‘gene expression-targeted isoflavone therapy’.

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.

Switch from θ to σ replication of bacteriophage λ DNA: factors involved in the process and a model for its regulation

Bacteriophage λ genome is one of the classical model replicons in studies on the regulation of DNA replication. Moreover, since genes coding for Shiga toxins are located in genomes of lambdoid phages, understanding of mechanisms controlling λ DNA replication may be of bio-medical importance. During lytic development of bacteriophage λ, its genome is replicated according to the θ (circle-to-circle) mode early after infection, and then it is switched to the σ (rolling circle) mode. Two mechanisms of regulation of this switch were proposed recently and both suggested a crucial role for directionality of λ DNA replication. Whereas one hypothesis assumed transient impairment of ClpP/ClpX-mediated proteolysis of the λO initiator protein, another suggested a crucial role for transcriptional activation of the oriλ region and factors involved in the control of the pR promoter activity. Here we demonstrate that mutations in clpP and clpX genes had little influence on both directionality of λ DNA replication and appearance of σ replication intermediates. On the other hand, regulators affecting activity of the pR promoter (responsible for initiation of transcription, which activates oriλ) directly or indirectly influenced directionality of λ DNA replication to various extents. Therefore, we conclude that regulation of the efficiency of transcriptional activation of oriλ, rather than transient impairment of the λO proteolysis, is responsible for the control of the switch from θ to σ replication, and propose a model for this control.

Interplay Between DnaA and SeqA Proteins During Regulation of Bacteriophage λ pR Promoter Activity

Abstract DnaA and SeqA proteins are main regulators (positive and negative, respectively) of the chromosome replication in Escherichia coli. Nevertheless, both these replication regulators were found recently to be also transcription factors. Interestingly, both DnaA and SeqA control activity of the bacteriophage λpR promoter by binding downstream of the transcription start site, which is unusual among prokaryotic systems. Here we asked what are functional relationships between these two transcription regulators at one promoter region. Both in vivo and in vitro studies revealed that DnaA and SeqA can activate the pR promoter independently and separately rather than in co-operation, however, increased concentrations of one of these proteins negatively influenced the transcription stimulation mediated by the second regulator. This may suggest a competition between DnaA and SeqA for binding to the pR regulatory region. The physiological significance of this DnaA and SeqA-mediated regulation of pR is demonstrated by studies on λ plasmid DNA replication in vivo.

Replicating DNA by cell factories: roles of central carbon metabolism and transcription in the control of DNA replication in microbes, and implications for understanding this process in human cells

Precise regulation of DNA replication is necessary to ensure the inheritance of genetic features by daughter cells after each cell division. Therefore, determining how the regulatory processes operate to control DNA replication is crucial to our understanding and application to biotechnological processes. Contrary to early concepts of DNA replication, it appears that this process is operated by large, stationary nucleoprotein complexes, called replication factories, rather than by single enzymes trafficking along template molecules. Recent discoveries indicated that in bacterial cells two processes, central carbon metabolism (CCM) and transcription, significantly and specifically influence the control of DNA replication of various replicons. The impact of these discoveries on our understanding of the regulation of DNA synthesis is discussed in this review. It appears that CCM may influence DNA replication by either action of specific metabolites or moonlighting activities of some enzymes involved in this metabolic pathway. The role of transcription in the control of DNA replication may arise from either topological changes in nucleic acids which accompany RNA synthesis or direct interactions between replication and transcription machineries. Due to intriguing similarities between some prokaryotic and eukaryotic regulatory systems, possible implications of studies on regulation of microbial DNA replication on understanding such a process occurring in human cells are discussed.

Bacteriophage and host mutants causing the rolling-circle λ DNA replication early after infection

There are two modes of bacteriophage λ DNA replication during its lytic development in Escherichia coli cells. The circle‐to‐circle (θ) replication predominates at early stages of the phage growth, whereas rolling‐circle (σ) replication occurs late after infection to produce long concatemers that serve as substrates for packaging of λ DNA into phage proheads. The mechanism regulating the switch from θ to σ replication remains unknown. Our previous genetic studies indicated that the bacteriophage λ Pts1πA66 mutant cannot replicate at 43°C in the wild‐type E. coli host, but it can replicate in the dnaA46(ts) mutant. Density shift experiments suggested that the parental DNA molecules of the infecting phage enter σ replication. Here, using electron microscopy, we demonstrate that as soon as 5 min after infection of the dnaA46(ts) mutant by the λPts1πA66 phage at 43°C, the σ replication intermediates are highly predominant over θ replication intermediates, contrary to the wild‐type conditions (wild‐type bacteria infected with the λP + phage). The initiation of replication of the λPts1πA66 mutant at 43°C was strongly inhibited in the dnaA + host, as demonstrated by electron microscopy and by pulse‐labeling of the phage‐derived plasmid replicon. Implications for the mechanism of the regulation of the switch from θ to σ replication mode are discussed.

Genes from the exo–xis region of λ and Shiga toxin-converting bacteriophages influence lysogenization and prophage induction

The exo–xis region, present in genomes of lambdoid bacteriophages, contains highly conserved genes of largely unknown functions. In this report, using bacteriophage λ and Shiga toxin-converting bacteriophage ϕ24Β, we demonstrate that the presence of this region on a multicopy plasmid results in impaired lysogenization of Escherichia coli and delayed, while more effective, induction of prophages following stimulation by various agents (mitomycin C, hydrogen peroxide, UV irradiation). Spontaneous induction of λ and ϕ24Β prophages was also more efficient in bacteria carrying additional copies of the corresponding exo–xis region on plasmids. No significant effects of an increased copy number of genes located between exo and xis on both efficiency of adsorption on the host cells and lytic development inside the host cell of these bacteriophages were found. We conclude that genes from the exo–xis region of lambdoid bacteriophages participate in the regulation of lysogenization and prophage maintenance.

Composition of the lambda plasmid heritable replication complex.

Previous studies indicated during replication of plasmids derived from bacteriophage lambda (the so-called lambda plasmids), that, once assembled, replication complex can be inherited by one of the two daughter plasmid copies after each replication round, and may function in subsequent replication rounds. It seems that similar processes occur during replication of other DNA molecules, including chromosomes of the yeast Saccharomyces cerevisiae. However, apart from some suggestions based on genetic experiments, composition of the lambda heritable replication complex remains unknown. In amino acid-starved Escherichia coli relA mutants, replication of lambda plasmid DNA is carried out exclusively by the heritable replication complex as assembly of new complexes is impaired due to inhibition of protein synthesis. Here, using a procedure based on in vivo cross-linking, cell lysis, immunoprecipitation with specific sera, de-cross-linking and PCR analysis, we demonstrate that the lambda heritable replication complex consists of O, P, DnaB and, perhaps surprisingly, DnaK proteins.

Mitochondrial DNA levels in Huntington disease leukocytes and dermal fibroblasts

Huntington disease (HD) is an inherited neurodegenerative disorder caused by mutations in the huntingtin gene. Involvement of mitochondrial dysfunctions in, and especially influence of the level of mitochondrial DNA (mtDNA) on, development of this disease is unclear. Here, samples of blood from 84 HD patients and 79 controls, and dermal fibroblasts from 10 HD patients and 9 controls were analysed for mtDNA levels. Although the type of mitochondrial haplogroup had no influence on the mtDNA level, and there was no correlation between mtDNA level in leukocytes in HD patients and various parameters of HD severity, some considerable differences between HD patients and controls were identified. The average mtDNA/nDNA relative copy number was significantly higher in leukocytes, but lower in fibroblasts, of symptomatic HD patients relative to the control group. Moreover, HD women displayed higher mtDNA levels in leukocytes than HD men. Because this is the largest population analysed to date, these results might contribute to explanation of discrepancies between previously published studies concerning levels of mtDNA in cells of HD patients. We suggest that the size of the investigated population and type of cells from which DNA is isolated could significantly affect results of mtDNA copy number estimation in HD. Hence, these parameters should be taken into consideration in studies on mtDNA in HD, and perhaps also in other diseases where mitochondrial dysfunction occurs.

A dual promoter system regulating λ DNA replication initiation.

Transcription and DNA replication are tightly regulated to ensure coordination of gene expression with growth conditions and faithful transmission of genetic material to progeny. A large body of evidence has accumulated, indicating that encounters between protein machineries carrying out DNA and RNA synthesis occur in vivo and may have important regulatory consequences. This feature may be exacerbated in the case of compact genomes, like the one of bacteriophage λ, used in our study. Transcription that starts at the rightward pR promoter and proceeds through the λ origin of replication and downstream of it was proven to stimulate the initiation of λ DNA replication. Here, we demonstrate that the activity of a convergently oriented pO promoter decreases the efficiency of transcription starting from pR. Our results show, however, that a lack of the functional pO promoter negatively influences λ phage and λ-derived plasmid replication. We present data, suggesting that this effect is evoked by the enhanced level of the pR-driven transcription, occurring in the presence of the defective pO, which may result in the impeded formation of the replication initiation complex. Our data suggest that the cross talk between the two promoters regulates λ DNA replication and coordinates transcription and replication processes.