Y-family DNA polymerases in mammalian cells

Eukaryotic genomes are replicated with high fidelity to assure the faithful transmission of genetic information from one generation to the next. The accuracy of replication relies heavily on the ability of replicative DNA polymerases to efficiently select correct nucleotides for the polymerization reaction and, using their intrinsic exonuclease activities, to excise mistakenly incorporated nucleotides. Cells also possess a variety of specialized DNA polymerases that, by a process called translesion DNA synthesis (TLS), help overcome replication blocks when unrepaired DNA lesions stall the replication machinery. This review considers the properties of the Y-family (a subset of specialized DNA polymerases) and their roles in modulating spontaneous and genotoxic-induced mutations in mammals. We also review recent insights into the molecular mechanisms that regulate PCNA monoubiquitination and DNA polymerase switching during TLS and discuss the potential of using Y-family DNA polymerases as novel targets for cancer prevention and therapy.     

Studies on DNA methylase activity in mammalian tissue

Resumen Para aislar nucleo de mamíferos que contienen DNA metilasa actividad se utilizó como medio homogenizante 0.25M sucrosa-1×10^–2 M EDTA. Hígado de rata y el hepatoma de Reuber demostraron cantidad equivalente de actividad enzimática. Se observó aproximadamente 100% de inhibición con amoníaco 0.15M, los iones de Mg no mostraron efecto.     

Homologous recombination-mediated repair of DNA double-strand breaks operates in mammalian mitochondria

Mitochondrial DNA is frequently exposed to oxidative damage, as compared to nuclear DNA. Previously, we have shown that while microhomology-mediated end joining can account for DNA deletions in mitochondria, classical nonhomologous DNA end joining, the predominant double-strand break (DSB) repair pathway in nucleus, is undetectable. In the present study, we investigated the presence of homologous recombination (HR) in mitochondria to maintain its genomic integrity. Biochemical studies revealed that HR-mediated repair of DSBs is more efficient in the mitochondria of testes as compared to that of brain, kidney and spleen. Interestingly, a significant increase in the efficiency of HR was observed when a DSB was introduced. Analyses of the clones suggest that most of the recombinants were generated through reciprocal exchange, while ~ 30% of recombinants were due to gene conversion in testicular extracts. Colocalization and immunoblotting studies showed the presence of RAD51 and MRN complex proteins in the mitochondria and immunodepletion of MRE11, RAD51 or NIBRIN suppressed the HR-mediated repair. Thus, our results reveal importance of homologous recombination in the maintenance of mitochondrial genome stability.     

Biochemistry of the erythrocyte

Zusammenfassung Der Erythrozyt hat zwei Hauptwege für den Glukoseabbau. Der Emden-Meyerhoff-Weg resultiert in der Phosphorylierung von ADP zu ATP und der Reduktion von NAD⁺ zu NADH. Der Hexose-Monophosphat-Weg ermöglicht andererseits die Reduktion von NADP⁺ zu NADPH. Es besteht eine beachtliche Beweglichkeit in der Kontrolle dieser Bahnen, die es dem Erythrozyten ermöglichen, seine Enzyme und sein Hämoglobin in aktiver Form und das Konzentrationsgefälle von Natrium und Kalium zwischen roten Blutkörperchen und Plasma zu erhalten.

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This work was made possible by a supporting fund established in the name of the Barry T. Leithead Research Fellowship, and was supported, in part, by Public Health Service Grant No. HE 07449 from the National Heart Institute, National Institutes of Health.     

Biochemistry of frog ribonucleases

Frogs have unique pyrimidine base-specific RNases, with structures similar to those of turtle, iguana and chicken RNases. Among the four frog RNases discussed here, three from Rana pipiens, R. catesbeiana and R. japonica oocyte cells show anti-tumour activity, and the latter two show lectin activity towards sialic acid-rich glycoproteins. In this review, (i) we compare their unique primary structures with respect to the locations of their disulphide bridges, three-dimensional structure, base specificity and heat stability as compared with RNase A, and (ii) we summarize current knowledge about the mode of action of lectin and the antitumour activities of the three frog RNases.     

Effect of pulsing electromagnetic fields on DNA synthesis in mammalian cells in culture

Summary DNA synthesis in Chinese hamster V79 cells was significantly enhanced when they were exposed to weak, pulsing electromagnetic fields generated by specific combinations of the pulse width (25s), frequency (10, 100 Hz) and magnetic intensity (2×10^–5 8×10^–5T). Conversely the DNA synthesis of cells in the fields at 4×10^–4 T was repressed to 80% of that in controls not exposed to the fields.This work was supported by Kaken Pharmaceutical Co., Ltd, Tokyo, Japan, and in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan and from the Foundation for Life Science Promotion Tokyo, Japan to I.K.     

Effect of pulsing electromagnetic fields on DNA synthesis in mammalian cells in culture

DNA synthesis in Chinese hamster V79 cells was significantly enhanced when they were exposed to weak, pulsing electromagnetic fields generated by specific combinations of the pulse width (25 microseconds), frequency (10, 100 Hz) and magnetic intensity (2 X 10(-5), 8 X 10(-5) T). Conversely the DNA synthesis of cells in the fields at 4 X 10(-4) T was repressed to 80% of that in controls not exposed to the fields.     

SET domain protein lysine methyltransferases: Structure, specificity and catalysis

Site- and state-specific lysine methylation of histones is catalyzed by a family of proteins that contain the evolutionarily conserved SET domain and plays a fundamental role in epigenetic regulation of gene activation and silencing in all eukaryotes. The recently determined three-dimensional structures of the SET domains from chromosomal proteins reveal that the core SET domain structure contains a two-domain architecture, consisting of a conserved anti-parallel β-barrel and a structurally variable insert that surround a unusual knot-like structure that comprises the enzyme active site. These structures of the SET domains, either in the free state or when bound to cofactor S-adenosyl-L-homocysteine and/or histone peptide, mimicking an enzyme/cofactor/substrate complex, further yield the structural insights into the molecular basis of the substrate specificity, methylation multiplicity and the catalytic mechanism of histone lysine methylation. Received 10 June 2006; accepted 22 August 2006     

Biochemistry of proinflammatory macrophage activation

In the last decade, metabolism has been recognized as a major determinant of immunological processes. During an inflammatory response, macrophages undergo striking changes in their metabolism. This metabolic reprogramming is governed by a complex interplay between metabolic enzymes and metabolites of different pathways and represents the basis for proper macrophage function. It is now evident that these changes go far beyond the well-known Warburg effect and the perturbation of metabolic targets is being investigated as a means to treat infections and auto-immune diseases. In the present review, we will aim to provide an overview of the metabolic responses during proinflammatory macrophage activation and show how these changes modulate the immune response.     

Partial complementation of a DNA ligase I deficiency by DNA ligase III and its impact on cell survival and telomere stability in mammalian cells

DNA ligase I (LigI) plays a central role in the joining of strand interruptions during replication and repair. In our current study, we provide evidence that DNA ligase III (LigIII) and XRCC1, which form a complex that functions in single-strand break repair, are required for the proliferation of mammalian LigI-depleted cells. We show from our data that in cells with either dysfunctional LigI activity or depleted of this enzyme, both LigIII and XRCC1 are retained on the chromatin and accumulate at replication foci. We also demonstrate that the LigI and LigIII proteins cooperate to inhibit sister chromatid exchanges but that only LigI prevents telomere sister fusions. Taken together, these results suggest that in cells with dysfunctional LigI, LigIII contributes to the ligation of replication intermediates but not to the prevention of telomeric instability.     

Biochemistry of the non-mevalonate isoprenoid pathway

The non-mevalonate pathway of isoprenoid (terpenoid) biosynthesis is essential in many eubacteria including the major human pathogen, Mycobacterium tuberculosis, in apicomplexan protozoa including the Plasmodium spp. causing malaria, and in the plastids of plants. The metabolic route is absent in humans and is therefore qualified as a promising target for new anti-infective drugs and herbicides. Biochemical and structural knowledge about all enzymes involved in the pathway established the basis for discovery and development of inhibitors by high-throughput screening of compound libraries and/or structure-based rational design.