Dependence of the viability and mutability of Escherichia coli K-12 cells on the balance of DNA and protein syntheses. II. The effect of disturbance in the balance of DNA and protein syntheses on the cellular mutability and viability of rec+ and rec- strains treated with nalidixic acid]

The changes in the survival and mutagenesis of rec+ and rec- Escherichia coli K-12 strains, treated with the selective inhibitor of DNA synthesis, nalidixic acid, are found to be due to the processes of the stabilization and repair of the metabolic gaps in DNA chains, which depend on the balance of DNA and protein synthesis. The various character is observed of the relation between the survival and the mutagenesis and the balance of DNA and protein syntheses in cells which are valuable (rec+) and defective (rec-) for the processes of DNA repair.     

Relation between Escherichia coli K-12 viability and mutability and the balance between DNA and protein synthesis. III. Relation between disruptions in the balance between DNA and protein synthesis and mutagenesis and viability during thymidine deprivation of thy- cells defective with respect to recB and polA genes

The phenomenon of metabolic mutagenesis is found to be determined by stabilization of metabolic breaks in DNA chains, being linked with disbalance of intracellular synthesis of DNA and protein. The rate of metabolic mutagenesis observed in case of the DNA-protein synthesis disbalance due to thymine starvation is influenced by cell genotype. The lack of exonuclease V in recB-thy- cells decreases (reduces) the rate of metabolic mutagenesis and does not effect the viability. The lack of DNA polymerase I activity in polA-thy- cells causes a sharp increase in the metabolic mutagenesis rate and a parallel sharp drop in the survival under thymine starvation, as compared to cells with polA+thy- genotype.     

The role of peroxisome proliferator activated receptors in metabolic balance disturbances under stress

Some aspects of peroxisome proliferator activated receptors (PPAR) involvement in regulation of stress-dependent biological processes leading to insulin resistance, lipid imbalance, hypertension and inflammation are reviewed. Analysis of literature data clearly shows the main role of PPAR in stress signal transduction following to metabolic disbalance development under prolonged stress conditions. The interplay of three PPAR isoforms functional activity with metabolic process disturbances during stress is under special emphasis. Taking into account experimental data described in literature we suggest that PPAR activation under acute stress is an adaptive response while stable PPAR hyperexpression under prolonged stress can cause insulin resistance, hypertension, and visceral obesity. The strategy of PPAR using as pharmacological targets in metabolic syndrome correction is under consideration.     

recF-dependent and recF recB-independent DNA gap-filling repair processes transfer dimer-containing parental strands to daughter strands in Escherichia coli K-12 uvrB.

The processes for repairing DNA daughter-strand gaps were studied in UV-irradiated uvrB, uvrB recB, uvrB recF, and uvrB recB recF cells of Escherichia coli K-12. The dimer-containing parental DNA was found to be joined to daughter strands during postreplication repair in all four strains examined. Therefore, both the major (recF-dependent) and the minor (recF recB-independent) gap-filling processes repair DNA daughter-strand gaps by transferring parental strands into daughter strands.     

Cis and trans mechanisms of DNA repair.

DNA repair is essential for genetic stability and variability. Remarkable advances in the understanding of DNA repair by the molecular analysis of the substrate (gene repair) or the enzyme (repair genes), emphasize evolutionary conservation. Recent progress also stresses the interaction(s) between DNA repair and numerous other cellular metabolic processes, including non-nuclear and/or non-genetic responses.     

Role of the radB gene in postreplication repair in UV-irradiated Escherichia coli uvrB

In UV-irradiated Escherichia coli, the radB101 mutation sensitized uvrB recF cells 4-fold and uvrB recB cells 1.2-fold, but did not sensitize uvrB recB recF cells. The radB mutation had very little effect (1.2-fold or less) on the repair of UV radiation-induced DNA daughter-strand gaps in uvrB cells, but it did cause about a 3-fold deficiency in the repair of the DNA double-strand breaks that arise in association with nonrepaired daughter-strand gaps in UV-irradiated uvrB recF cells. Thus, the radB gene does not appear to be involved in the recF-dependent or recF recB-independent processes for the repair of DNA daughter-strand gaps, but is involved in the recB-dependent postreplication repair of DNA double-strand breaks.     

Nutrition--a factor in preventing arteriosclerosis, coronary disease and disorders of the blood coagulation system. I. An imbalance in food fats and carbohydrates and atherogenesis

The analysis of the literary data and the results of own research testifies that the lipids and carbohydrates metabolism disturbance in food is one of the decisive factors of pathogenetic arteriosclerosis and coronary disease. A long balanced diet in particular in fats and carbohydrates favours the normalization of the metabolic processes in stabilization of the organism.     

Microbial anhydrobiosis

The loss of cellular water (desiccation) and the resulting low cytosolic water activity are major stress factors for life. Numerous prokaryotic and eukaryotic taxa have evolved molecular and physiological adaptions to periods of low water availability or water-limited environments that occur across the terrestrial Earth. The changes within cells during the processes of desiccation and rehydration, from the activation (and inactivation) of biosynthetic pathways to the accumulation of compatible solutes, have been studied in considerable detail. However, relatively little is known on the metabolic status of organisms in the desiccated state; that is, in the sometimes extended periods between the drying and rewetting phases. During these periods, which can extend beyond decades and which we term ‘anhydrobiosis’, organismal survival could be dependent on a continued supply of energy to maintain the basal metabolic processes necessary for critical functions such as macromolecular repair. Here, we review the state of knowledge relating to the function of microorganisms during the anhydrobiotic state, highlighting substantial gaps in our understanding of qualitative and quantitative aspects of molecular and biochemical processes in desiccated cells.     

Bacterial DNA repair genes and their eukaryotic homologues: 5. The role of recombination in DNA repair and genome stability

Recombinational repair is a well conserved DNA repair mechanism present in all living organisms. Repair by homologous recombination is generally accurate as it uses undamaged homologous DNA molecule as a repair template. In Escherichia coli homologous recombination repairs both the double-strand breaks and single-strand gaps in DNA. DNA double-strand breaks (DSB) can be induced upon exposure to exogenous sources such as ionizing radiation or endogenous DNA-damaging agents including reactive oxygen species (ROS) as well as during natural biological processes like conjugation. However, the bulk of double strand breaks are formed during replication fork collapse encountering an unrepaired single strand gap in DNA. Under such circumstances DNA replication on the damaged template can be resumed only if supported by homologous recombination. This functional cooperation of homologous recombination with replication machinery enables successful completion of genome duplication and faithful transmission of genetic material to a daughter cell. In eukaryotes, homologous recombination is also involved in essential biological processes such as preservation of genome integrity, DNA damage checkpoint activation, DNA damage repair, DNA replication, mating type switching, transposition, immune system development and meiosis. When unregulated, recombination can lead to genome instability and carcinogenesis.     

p53 is a rate-limiting factor in the repair of higher-order DNA structure.

The product of the p53 tumor suppressor gene has been implicated in safeguarding genomic stability by transactivating genes involved in cell cycle arrest, repair of DNA damage or induction of apoptosis. Several properties of p53 suggest that it might be directly involved in DNA repair processes. Eukaryotic DNA is highly organized in supercoiled loops anchored to the nuclear matrix. This organization is very important for cell function and survival, suggesting that repair of DNA damage must include both, the integrity of the double helix and the complex DNA topology. In this work, we studied the kinetics and efficiency of higher-order DNA structure repair in cells with normal and reduced levels of p53, and present evidence suggesting that p53 may be involved in the stabilization and/or repair of higher-order DNA structure.     

Circadian regulation and its disorders in Parkinson’s disease patients. Part 1: The role of dopamine in circadian dysfunction

Circadian disturbances related to Parkinson’s disease are reviewed, and possible pathogenetic mechanisms are discussed. The role of dopaminergic system degeneration in the development of circadian dysfunction is emphasized. The accumulation of α-synuclein in the suprachiasmatic nucleus is considered as a possible mechanism of circadian dysfunction unrelated to dopamine deficiency. Data on the disbalance of dopamine and melatonin levels in Parkinson’s disease patients and its role in disturbances of circadian rhythms of physiological processes are analyzed.     

Archaeal DNA replication and repair

Since the first archaeal genome was sequenced, much attention has been focused on the study of these unique microorganisms. We have learnt that although archaeal DNA metabolic processes (replication, recombination and repair) are more similar to the metabolic processes of Eukarya than those of Bacteria, Archaea are not simply 'mini Eukarya'. They are, in fact, a mosaic of the eukaryal and bacterial systems that also possess archaeal-specific features. Recent biochemical and structural studies of the proteins that participate in archaeal DNA replication and repair have increased our understanding of these processes.     

Stabilization of the tumor suppressor p53 during cellular transformation by simian virus 40: influence of viral and cellular factors and biological consequences.

To understand the process and biological significance of metabolic stabilization of p53 during simian virus 40 (SV40)-induced cellular transformation, we analyzed cellular and viral parameters involved in this process. We demonstrate that neither large T expression as such nor the cellular phenotype (normal versus transformed) markedly influence the stability of p53 complexed to large T in SV40 abortively infected BALB/c mouse fibroblasts. In contrast, metabolic stabilization of p53 is an active cellular event, specifically induced by SV40. The ability of SV40 to induce a cellular response leading to stabilization of p53 complexed to large T is independent from the cellular phenotype and greatly varies between different cells. However, metabolic stability was conferred only to p53 in complex with large T, whereas the free p53 in these cells remained metabolically unstable. Comparative analyses of cellular transformation in various cells differing in stability of p53 complexed to large T upon abortive infection with SV40 revealed a strong correlation between the ability of SV40 to induce metabolic stabilization and its transformation efficiency. Our data suggest that metabolic stabilization and the ensuing enhanced levels of p53 are important for initiation and/or maintenance of SV40 transformation.     

Different effects of recJ and recN mutations on the postreplication repair of UV-damaged DNA in Escherichia coli K-12.

Two mutations known to affect recombination in a recB recC sbsBC strain, recJ284::Tn10 and recN262, were examined for their effects on the postreplication repair of UV-damaged DNA. The recJ mutation did not affect the UV radiation sensitivity of uvrB and uvrB recF cells, but it increased the sensitivity of uvrB recN (approximately 3-fold) and uvrB recB (approximately 8-fold) cells. On the other hand, the recN mutation did not affect the UV sensitivity of uvrB recB cells, but it increased the sensitivity of uvrB (approximately 1.5-fold) and uvrB recF (approximately 4-fold) cells. DNA repair studies indicated that the recN mutation produced a partial deficiency in the postreplication repair of DNA double-strand breaks that arise from unrepaired daughter strand gaps, while the recJ mutation produced a deficiency in the repair of daughter strand gaps in uvrB recB cells (but not in uvrB cells) and a deficiency in the repair of both daughter strand gaps and double-strand breaks in uvrA recB recC shcBC cells. Together, these results indicate that the recJ and recN genes are involved in different aspects of postreplication repair.     

Postreplication repair of DNA in chick cells: studies using photoreactivation.

During replication of DNA after ultraviolet irradiation, gaps are left in the newly-synthesized DNA strands in both bacterial and animal cells and these gaps are subsequently sealed by a process known as postreplication repair. In order to test whether it is the ultraviolet-induced pyrimidine dimers which are responsible for the production of these daughter-strand gaps in animal cells, we have used chick embryo fibroblasts. In these cells the pyrimidine dimers are photoreactivable, i.e. they can be split by an enzymatic process dependent on visible or near ultraviolet light. Our results indicate that chick cells possess a postreplication repair system similar to that in mammalian cells; gaps are produced in the newly-synthesized strands and then filled in. If the ultraviolet-irradiated cells are first photoreactivated to remove most of the dimers, the number of daughter-strand gaps produced is much less than without photoreactivation. This suggests that the dimers are indeed responsible for the formation of many of the gaps in the newly-synthesized DNA. Ultraviolet light also inhibits the overall rate of DNA synthesis. This inhibition is, however, only partly overcome by photoreactivation.     

Metabolite damage and repair in metabolic engineering design

The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields, and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways – particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile ‘plug and play’ set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects.     

Mechanisms for recF-dependent and recB-dependent pathways of postreplication repair in UV-irradiated Escherichia coli uvrB.

The molecular mechanisms for the recF-dependent and recB-dependent pathways of postreplication repair were studied by sedimentation analysis of DNA from UV-irradiated Escherichia coli cells. When the ability to repair DNA daughter strand gaps was compared, uvrB recF cells showed a gross deficiency, whereas uvrB recB cells showed only a small deficiency. Nevertheless, the uvrB recF cells were able to perform some limited repair of daughter strand gaps compared with a "repairless" uvrB recA strain. The introduction of a recB mutation into the uvrB recF strain greatly increased its UV radiation sensitivity, yet decreased only slightly its ability to repair daughter strand gaps. Kinetic studies of DNA repair with alkaline and neutral sucrose gradients indicated that the accumulation of unrepaired daughter strand gaps led to the formation of low-molecular-weight DNA duplexes (i.e., DNA double-strand breaks were formed). The uvrB recF cells were able to regenerate high-molecular-weight DNA from these low-molecular-weight DNA duplexes, whereas the uvrB recF recB and uvrB recA cells were not. A model for the recB-dependent pathway of postreplication repair is presented.     

The involvement of polynucleotide ligase in the repair of UV-induced DNA damage in Escherichia coli K-12 cells.

Summary The effect of the ligts-7 mutation on cell survival and the extent of DNA repair after UV (254 nm) irradiation was determined for wild-type and uvrB5 cells of E. coli K-12 at 30° and 42°C. At the restrictive temperature (42°C) the ligts-7 mutation resulted in (i) a decrease in the extent of repair of DNA incision breaks arising during the excision repair process, and (ii) a decrease in the extent of post-replicational repair of gaps in newly-synthesized DNA. These deficiencies in DNA repair correlated with increases in cellular sensitivity to killing by UV radiation. Thus, DNA ligase plays an important role in vivo in both the excision and post-replicational repair processes.     

Cooperation of simian virus 40 large and small T antigens in metabolic stabilization of tumor suppressor p53 during cellular transformation.

Metabolic stabilization of the tumor suppressor p53 is a key event in cellular transformation by simian virus 40 (SV40). Expression of the SV40 large tumor antigen (large T) is necessary but not sufficient for this process, as metabolic stabilization of p53 complexed to large T in abortively SV40-infected cells strictly depends on the cellular systems analyzed (F. Tiemann and W. Deppert, J. Virol. 68:2869-2878, 1994). Comparative analyses of various cells differing in metabolic stabilization of p53 upon abortive infection with SV40 revealed that metabolic stabilization of p53 closely correlated with expression of the SV40 small t antigen (small t) in these cells: 3T3 cells do not express small t and do not stabilize p53 upon infection with wild-type SV40. However, ectopic expression of small t in 3T3 cells provided these cells with the capacity to stabilize p53 upon SV40 infection. Conversely, precrisis mouse embryo cells express small t and mediate metabolic stabilization of p53 upon infection with wild-type SV40. Infection of these cells with an SV40 small-t deletion mutant did not lead to metabolic stabilization of p53. Small-t expression and metabolic stabilization of p53 correlated with an enhanced transformation efficiency by SV40, supporting the conclusion that at least part of the documented helper effect of small t in SV40 transformation is its ability to promote metabolic stabilization of p53 complexed to large T.     

DNA damage induced hyperphosphorylation of replication protein A. 2. Characterization of DNA binding activity, protein interactions, and activity in DNA replication and repair

Replication protein A (RPA) is a heterotrimeric protein consisting of 70-, 34-, and 14- kDa subunits that is required for many DNA metabolic processes including DNA replication and DNA repair. Using a purified hyperphosphorylated form of RPA protein prepared in vitro, we have addressed the effects of hyperphosphorylation on steady-state and pre-steady-state DNA binding activity, the ability to support DNA repair and replication reactions, and the effect on the interaction with partner proteins. Equilibrium DNA binding activity measured by fluorescence polarization reveals no difference in ssDNA binding to pyrimidine-rich DNA sequences. However, RPA hyperphosphorylation results in a decreased affinity for purine-rich ssDNA and duplex DNA substrates. Pre-steady-state kinetic analysis is consistent with the equilibrium DNA binding and demonstrates a contribution from both the k(on) and k(off) to achieve these differences. The hyperphosphorylated form of RPA retains damage-specific DNA binding, and, importantly, the affinity of hyperphosphorylated RPA for damaged duplex DNA is 3-fold greater than the affinity of unmodified RPA for undamaged duplex DNA. The ability of hyperphosphorylated RPA to support DNA repair showed minor differences in the ability to support nucleotide excision repair (NER). Interestingly, under reaction conditions in which RPA is maintained in a hyperphosphorylated form, we also observed inhibition of in vitro DNA replication. Analyses of protein-protein interactions bear out the effects of hyperphosphorylated RPA on DNA metabolic pathways. Specifically, phosphorylation of RPA disrupts the interaction with DNA polymerase alpha but has no significant effect on the interaction with XPA. These results demonstrate that the effects of DNA damage induced hyperphosphorylation of RPA on DNA replication and DNA repair are mediated through alterations in DNA binding activity and protein-protein interactions.