Robert Łyżeń

ppGpp inhibits the activity of Escherichia coli DnaG primase

DNA primase is an enzyme required for replication of both chromosomes and vast majority of plasmids. Guanosine tetra- and penta-phosphate (ppGpp and pppGpp, respectively) are alarmones of the bacterial stringent response to starvation and stress conditions, and act by modulation of the RNA polymerase activity. Recent studies indicated that the primase-catalyzed reaction is also inhibited by (p)ppGpp in Bacillus subtilis, where a specific regulation of DNA replication elongation, the replication fork arrest, was discovered. Although in Escherichia coli such a replication regulation was not reported to date, here we show that E. coli DnaG primase is directly inhibited by ppGpp and pppGpp. However, contrary to the B. subtilis primase response to the stringent control alarmones, the E, coli DnaG was inhibited more efficiently by ppGpp than by pppGpp.

Transcription from bacteriophage λ pR promoter is regulated independently and antagonistically by DksA and ppGpp

The stringent response effector, guanosine tetraphosphate (ppGpp), adjust gene expression and physiology in bacteria, by affecting the activity of various promoters. RNA polymerase-interacting protein, DksA, was proposed to be the co-factor of ppGpp effects; however, there are reports suggesting independent roles of these regulators. Bacteriophage λ major lytic promoter, pR, is down-regulated by the stringent response and ppGpp. Here, we present evidence that DksA significantly stimulates pR-initiated transcription in vitro in the reconstituted system. DksA is also indispensable for pR activity in vivo. DksA-mediated activation of pR-initiated transcription is predominant over ppGpp effects in the presence of both regulators in vitro. The possible role of the opposite regulation by ppGpp and DksA in λ phage development is discussed. The major mechanism of DksA-mediated activation of transcription from pR involves facilitating of RNA polymerase binding to the promoter region, which results in more productive transcription initiation. Thus, our results provide evidence for the first promoter inhibited by ppGpp that can be stimulated by the DksA protein both in vivo and in vitro. Therefore, DksA role could be not only independent but antagonistic to ppGpp in transcription regulation.

Sensitivity of dark mutants of various strains of luminescent bacteria to reactive oxygen species

Recent studies indicated that bioluminescence of the marine bacterium Vibrio harveyi may both stimulate DNA repair and contribute to detoxification of deleterious oxygen derivatives. Therefore, it was also proposed that these reactions can be considered biological roles of bacterial luminescence and might act as evolutionary drives in development of luminous systems. However, experimental evidence for the physiological role of luciferase in protection of cells against oxidative stress has been demonstrated only in one bacterial species, raising the question whether this is a specific or a more general phenomenon. Here we demonstrate that in the presence of various oxidants (hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and ferrous ions) growth of dark mutants of different strains of Vibrio fischeri and Photobacterium leiognathi is impaired relative to wild-type bacteria, though to various extents. Deleterious effects of oxidants on the mutants could be reduced (with different efficiency) by addition of antioxidants, A-TEMPO or 4OH-TEMPO. These results support the hypotheses that (1) activities of bacterial luciferases may detoxify deleterious oxygen derivatives, and (2) significantly different efficiencies of this reaction are characteristic for various luciferases.

Effects of partial silencing of genes coding for enzymes involved in glycolysis and tricarboxylic acid cycle on the enterance of human fibroblasts to the S phase

BackgroundPreviously published reports indicated that some enzymes of the central carbon metabolism (CCM), particularly those involved in glycolysis and the tricarboxylic acid cycle, may contribute to regulation of DNA replication. However, vast majority of such works was performed with the use of cancer cells, in the light of carcinogenesis. On the other hand, recent experiments conducted on bacterial models provided evidence for the direct genetic link between CCM and DNA replication. Therefore, we asked if silencing of genes coding for glycolytic and/or Krebs cycle enzymes may affect the control of DNA replication in normal human fibroblasts.ResultsParticular genes coding for these enzymes were partially silenced with specific siRNAs. Such cells remained viable. We found that silencing of certain genes resulted in either less efficient or delayed enterance to the S phase. This concerned following genes: HK2, PFKM, TPI, GAPDH, ENO1, LDHA, CS1, ACO2, SUCLG2, SDHA, FH and MDH2. Decreased levels of expression of HK2, GADPH, CS1, ACO2, FH and MDH2 caused also a substantial impairment in DNA synthesis efficiency.ConclusionsThe presented results illustrate the complexity of the influence of genes coding for enzymes of glycolysis and the tricarboxylic acid cycle on the control of DNA replication in human fibroblasts, and indicate which of them are especially important in this process.

Enzymes of the central carbon metabolism: Are they linkers between transcription, DNA replication, and carcinogenesis?

Dependence of carcinogenesis on disruption of DNA replication regulation is a well-known fact. There are also many reports demonstrating the interplay between transcription and DNA replication processes, particularly underlying the importance of promoter activities in the control of replication initiation. Recent findings have shown direct links between central carbon metabolism and DNA replication regulation. Here, we summarize previously published reports which indicated that enzymes of the central carbon metabolism, particularly those involved in glycolysis and the tricarboxylic acid cycle, may contribute to regulation of transcription and DNA transactions (replication and repair). In this light, we propose a hypothesis that some of these enzymes might be linkers between transcription, DNA replication, and carcinogenesis. If true, it may have a consequence in our understanding of causes and mechanisms of carcinogenesis. Particularly, certain metabolic perturbations might directly (through central carbon metabolism enzymes) influence regulation of DNA transactions (replication control and fidelity), and thus facilitate carcinogenesis. To test this hypothesis, further studies will be necessary, which is discussed in the final part of this article.

Transcription regulation of the Escherichia coli pcnB gene coding for poly(A) polymerase I: roles of ppGpp, DksA and sigma factors

Poly(A) polymerase I (PAP I), encoded by the pcnB gene, is a major enzyme responsible for RNA polyadenylation in Escherichia coli, a process involved in the global control of gene expression in this bacterium through influencing the rate of transcript degradation. Recent studies have suggested a complicated regulation of pcnB expression, including a complex promoter region, a control at the level of translation initiation and dependence on bacterial growth rate. In this report, studies on transcription regulation of the pcnB gene are described. Results of in vivo and in vitro experiments indicated that (a) there are three σ70-dependent (p1, pB, and p2) and two σS-dependent (pS1 and pS2) promoters of the pcnB gene, (b) guanosine tetraphosphate (ppGpp) and DksA directly inhibit transcription from pB, pS1 and pS2, and (c) pB activity is drastically impaired at the stationary phase of growth. These results indicate that regulation of the pcnB gene transcription is a complex process, which involves several factors acting to ensure precise control of PAP I production. Moreover, inhibition of activities of pS1 and pS2 by ppGpp and DksA suggests that regulation of transcription from promoters requiring alternative σ factors by these effectors of the stringent response might occur according to both passive and active models.

Silencing of the pentose phosphate pathway genes influences DNA replication in human fibroblasts

Previous reports and our recently published data indicated that some enzymes of glycolysis and the tricarboxylic acid cycle can affect the genome replication process by changing either the efficiency or timing of DNA synthesis in human normal cells. Both these pathways are connected with the pentose phosphate pathway (PPP pathway). The PPP pathway supports cell growth by generating energy and precursors for nucleotides and amino acids. Therefore, we asked if silencing of genes coding for enzymes involved in the pentose phosphate pathway may also affect the control of DNA replication in human fibroblasts. Particular genes coding for PPP pathway enzymes were partially silenced with specific siRNAs. Such cells remained viable. We found that silencing of the H6PD, PRPS1, RPE genes caused less efficient enterance to the S phase and decrease in efficiency of DNA synthesis. On the other hand, in cells treated with siRNA against G6PD, RBKS and TALDO genes, the fraction of cells entering the S phase was increased. However, only in the case of G6PD and TALDO, the ratio of BrdU incorporation to DNA was significantly changed. The presented results together with our previously published studies illustrate the complexity of the influence of genes coding for central carbon metabolism on the control of DNA replication in human fibroblasts, and indicate which of them are especially important in this process.

The dual role of DksA protein in the regulation of Escherichia coli pArgX promoter

Gene expression regulation by the stringent response effector, ppGpp, is facilitated by DksA protein; however DksA and ppGpp can play independent roles in transcription. In Escherichia coli, the pArgX promoter which initiates the transcription of four tRNA genes was shown to be inhibited by ppGpp. Our studies on the role of DksA in pArgX regulation revealed that it can stimulate transcription by increasing the binding of RNA polymerase to the promoter and the productive transcription complex formation. However, when DksA is present together with ppGpp a severe down-regulation of promoter activity is observed. Our results indicate that DksA facilitates the effects of ppGpp to drive formation of inactive dead-end complexes formed by RNA polymerase at the ArgX promoter. In vivo, ppGpp-mediated regulation of pArgX transcription is dependent on DksA activity. The potential mechanisms of opposing pArgX regulation by ppGpp and DksA are discussed. pArgX is the first reported example of the promoter stimulated by DksA and inhibited by ppGpp in vitro when an overall inhibition occurs in the presence of both regulators. A dual role is thus proposed for DksA in the regulation of the pArgX promoter activity.

Characterization of the transcriptional stimulatory properties of the Pseudomonas putida RapA protein

RNA polymerase-associated factors can significantly affect its performance at specific promoters. Here we identified a Pseudomonas putida RNA polymerases-associated protein as a homolog of Escherichia coli RapA. We found that P. putida RapA stimulates the transcription from promoters dependent on a variety of σ-factors (σ(70), σ(S), σ(54), σ(32), σ(E)) in vitro. The level of stimulation varied from 2- to 10-fold, with the maximal effect observed with the σ(E)-dependent PhtrA promoter. Stimulation by RapA was apparent in the multi-round reactions and was modulated by salt concentration in vitro. However, in contrast to findings with E. coli RapA, P. putida RapA-mediated stimulation of transcription was also evident using linear templates. These properties of P. putida RapA were apparent using either E. coli- or P. putida-derived RNA polymerases. Analysis of individual steps of transcription revealed that P. putida RapA enhances the stability of competitor-resistant open-complexes formed by RNA polymerase at promoters. In vivo, P. putida RapA can complement the inhibitory effect of high salt on growth of an E. coli RapA null strain. However, a P. putida RapA null mutant was not sensitive to high salt. The in vivo effects of lack of RapA were only detectable for the σ(E)-PhtrA promoter where the RapA-deficiency resulted in lower activity. The presented characteristics of P. putida RapA indicate that its functions may extend beyond a role in facilitating RNA polymerase recycling to include a role in transcription initiation efficiency.

Double silencing of relevant genes suggests the existence of the direct link between DNA replication/repair and central carbon metabolism in human fibroblasts

Genetic evidence for a link between DNA replication and glycolysis has been demonstrated a decade ago in Bacillus subtilis, where temperature-sensitive mutations in genes coding for replication proteins could be suppressed by mutations in genes of glycolytic enzymes. Then, a strong influence of dysfunctions of particular enzymes from the central carbon metabolism (CCM) on DNA replication and repair in Escherichia coli was reported. Therefore, we asked if such a link occurs only in bacteria or it is a more general phenomenon. Here, we demonstrate that effects of silencing (provoked by siRNA) of expression of genes coding for proteins involved in DNA replication and repair (primase, DNA polymerase ι, ligase IV, and topoisomerase IIIβ) on these processes (less efficient entry into the S phase of the cell cycle and decreased level of DNA synthesis) could be suppressed by silencing of specific genes of enzymes from CMM. Silencing of other pairs of replication/repair and CMM genes resulted in enhancement of the negative effects of lower expression levels of replication/repair genes. We suggest that these results may be proposed as a genetic evidence for the link between DNA replication/repair and CMM in human cells, indicating that it is a common biological phenomenon, occurring from bacteria to humans.