Inhibitory effects of vitamin K₃ derivatives on DNA polymerase and inflammatory activity

Previously, we reported that vitamin K? (menadione, 2-methyl-1,4-naphthoquinone) (compound 2) inhibits the activity of human mitochondrial DNA polymerase γ (pol γ). In this study, we investigated the inhibitory effects (IEs) of vitamin K3 and its derivatives, such as 1,4-naphthoquinone (compound 1) and 1,2-dimethyl-1,4-naphthoquinone (compound 3), on the activity of mammalian pols. Among compounds 1-3 (10 μM for each), compound 1 was the strongest inhibitor of mammalian pols α and λ, which belong to the B and X pol families, respectively, whereas compound 2 was the strongest inhibitor of human pol γ, a family A pol. However, these compounds did not affect the activity of human pol κ, a family Y pol. As we previously found a positive relationship between pol λ inhibition and anti-inflammatory action, we examined whether these vitamin K? derivatives are able to inhibit inflammatory responses. Among the three compounds tested, compound 1 caused the greatest reduction in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced acute inflammation in mouse ears. In addition, in a cell culture system using RAW264.7 mouse macrophages, compound 1 displayed the strongest suppression of tumor necrosis factor (TNF)-α production induced by lipopolysaccharide (LPS). In an in vivo mouse model of LPS-evoked acute inflammation, the intraperitoneal injection of compound 1 into mice suppressed TNF-α production in their peritoneal macrophages and serum. In an in vivo colitis mouse model induced using dextran sulfate sodium (DSS), the vitamin K? derivatives markedly suppressed DSS-evoked colitis. In conclusion, this study has identified several vitamin K? derivatives, such as compound 1, that are promising anti-inflammatory candidates.     

Age-related changes in expression and activity of DNA polymerase α: some effects of dietary restriction

DNA polymerase α (pol α) purified from human diploid fibroblasts (HDF) and from livers of C57BL/6N mice showed age-related decreases in: (1) mRNA levels; (2) the amount of enzyme isolated per cell; and (3) enzyme activity (HDF); as well as: a) the amount of enzyme isolated; b) the specific activity; and c) the enzyme fidelity (liver). Hepatic pol α from dietary restricted (DR) mice exhibited less of a decline in specific activity and copied synthetic DNA templates with relatively higher fidelity than did enzymes from animals fed ad libitum (AL). Pol α from fetal-derived HDF exhibited increased expression compared with aged donor-derived HDF, with both fetal and old cell pol α in normal cells being expressed at lower levels than in their transformed cell corollaries. Treatment of human pol α from aged donor-derived HDF with a pol α accessory protein isolated from log phase murine cells resulted in increased pol α binding of DNA and increased pol α activity. However, highly active pol α isolated from fetal-derived or transformed HDF, or from transformed murine cells, showed little or no activity enhancement in the presence of accessory protein. These data indicate that, as a function of increased age, there is a decrease in pol α expression and specific activity in HDF, as well as decreases in specific activity and fidelity of pol α in essentially amitotic murine hepatic tissues. Dietary restriction impedes the age-related declines in both activity and fidelity of hepatic pol α in mice. The data further indicate that transformation of slowly dividing HDF is associated with increased expression of pol α, but suggest that increased expression alone is not sufficient to explain the difference in polymerase activity levels between parental and transformed HDF. Lastly, the data suggest that interaction of pol α with an essential accessory protein may be altered as a function of age, an alteration that appears to be correlated with the decline in pol α DNA binding and specific activity.     

Sequence-specific stalling of DNA polymerase γ and the effects of mutations causing progressive ophthalmoplegia

A large number of mutations in the gene encoding the catalytic subunit of mitochondrial DNA polymerase γ (POLγA) cause human disease. The Y955C mutation is common and leads to a dominant disease with progressive external ophthalmoplegia and other symptoms. The biochemical effect of the Y955C mutation has been extensively studied and it has been reported to lower enzyme processivity due to decreased capacity to utilize dNTPs. However, it is unclear why this biochemical defect leads to a dominant disease. Consistent with previous reports, we show here that the POLγA:Y955C enzyme only synthesizes short DNA products at dNTP concentrations that are sufficient for proper function of wild-type POLγA. In addition, we find that this phenotype is overcome by increasing the dNTP concentration, e.g. dATP. At low dATP concentrations, the POLγA:Y955C enzyme stalls at dATP insertion sites and instead enters a polymerase/exonuclease idling mode. The POLγA:Y955C enzyme will compete with wild-type POLγA for primer utilization, and this will result in a heterogeneous population of short and long DNA replication products. In addition, there is a possibility that POLγA:Y955C is recruited to nicks of mtDNA and there enters an idling mode preventing ligation. Our results provide a novel explanation for the dominant mtDNA replication phenotypes seen in patients harboring the Y955C mutation, including the existence of site-specific stalling. Our data may also explain why mutations that disturb dATP pools can be especially deleterious for mtDNA synthesis.     

DNA polymerase δ of Physarum polycephalum

DNA polymerase from the phylogenetically ancient slime mold Physarum polycephalum has been 380-fold enriched from amoebae. It was found to have the properties typical for this type of DNA polymerase from higher eukaryotes with regard to effectors, template-primer acceptance, co-purification with 3–5-exonuclease activity, as well as the effect of endogenous proliferating cell nuclear antigen (PCNA) from amoebae on the stimulation and processivity of DNA synthesis. An identified cDNA fragment shows 65.5% identical amino acides with DNA polymerase from Saccharomyces pombe. The molecular mass of the polymerase is 125 kDa while that of PCNA is 35 kDa. During size-exclusion chromatography, the highly purified polymerase eluted in the position of 125 kDa, suggesting that no other proteins were tightly complexed with the enzyme. The DNA polymerases from the (mononucleate) amoebae and from the (multinucleate) plasmodia of P. polycephalum have very similar properties in contrast to their differences in phenotype and their mode of nuclear division. The polymerase shows a higher degree of similarity than DNA polymerase , and especially the -like DNA polymerase, with the corresponding polymerases of higher eukaryotes. According to antibody staining, DNA polymerase is readily fragmented by proteases, even in the presence of inhibitor cocktails. Including freshly prepared cell lysates, proteolytic fragments are reproducible, the most abundant being 50 kDa in size. The DNA polymerase is recognized by the antisera against two peptides which have been derived by PCR-screening of plasmodial cDNA. One of the proteolytic splitting sites is located within an eight amino-acid stretch between the two antigenic sequences.     

An accessory protein enhances both DNA binding and activity of DNA polymerase α isolated from normal,but not transformed,human fibroblasts

DNA polymerase α/primase (pol α) isolated from fibroblasts established from a 66-year-old human donor (GM3529) exhibited decreased specific activity compared with pol α from either fetal-derived fibroblasts (W138), or pSV3.neo-transformed GM3529 fibroblasts. The pol α specific activity decrease was correlated with a decreased proliferative capacity frequently seen in cells from aged donors. Pol α isolated from pSV3.neo-transformed GM3529 cells (GM3529T) exhibited a single isoform with about 10-fold higher specific activity than pol α from GM3529 cells. GM3529T pol α was immunoreactive with both anti-pol α and anti-SV40 large tumor antigen. Polymerases from GM3529 and GM3529T cells were treated with a pol α accessory protein, αAP, isolated from L1210 cells. Pol α from GM3529T cells showed no increase in activity in the presence of αAP, while pol α isolated from GM3529 cells exhibited about an 8-fold increase in activity after treatment with αAP. Double stranded SV40 DNA containing multiple ori sequences exhibited a greater decrease in electrophoretic mobility in the presence of GM3529T pol α. In the presence of pol α from either GM35229 or GM3529T cells SV40 dsDNA exhibited a decrease in electrophoretic mobility, and in each instance addition of αAP resulted in an even greater decrease in DNAm mobility. These data indicate that αAP increased pol α binding to SV40 dsDNA, or that αAP bound the DNA in addition to previously bound pol α. GM3529 pol α also bound non-specific, non-SV40, dsDNA, whereas GM3529T pol α with associated TAg did not bind the non-viral dsDNA unless αAP was added to the preparation. While not all human diploid fibroblast cell lines derived from aged human donors necessarily exhibit decreased proliferative capacity compared with cells from young donors, decreased specific activity associated with a decline in cellular DNA synthesis is typical of pol α from cells derived from aged human donors. We suggest that a decrease in endogenous αAP interaction with pol α may account, in part, for the loss of DNA binding affinity and specific activity of pol α from GM3529 cells derived from an aged donor.     

Importin α2 association with chromatin: Direct DNA binding via a novel DNA-binding domain

The nuclear transport of proteins is important for facilitating appropriate nuclear functions. The importin α family proteins play key roles in nuclear transport as transport receptors for copious nuclear proteins. Additionally, these proteins possess other functions, including chromatin association and gene regulation. However, these nontransport functions of importin α are not yet fully understood, especially their molecular-level mechanisms and consequences for functioning with chromatin. Here, we report the novel molecular characteristics of importin α binding to diverse DNA sequences in chromatin. We newly identified and characterized a DNA-binding domain—the Nucleic Acid Associating Trolley pole domain (NAAT domain)—in the N-terminal region of importin α within the conventional importin β binding (IBB) domain that is necessary for nuclear transport of cargo proteins. Furthermore, we found that the DNA binding of importin α synergistically coupled the recruitment of its cargo protein to DNA. This is the first study to delineate the interaction between importin α and chromatin DNA via the NAAT domain, indicating the bifunctionality of the importin α N-terminal region for nuclear transport and chromatin association.     

Immunity and autoimmunity to dsDNA and chromatin – the role of immunogenic DNA-binding proteins and nuclease deficiencies

Loss of immunological tolerance results in autoimmunity and may finally end in autoimmune disorders. For an autoimmune response against chromatin, autologous chromatin (nucleosomes) is assumed to activate both chromatin-specific B and T cells with a resulting anti-chromatin antibody response. As only fragmental elements of this process have been described, we do not have the full insight to justify this model in vivo. Early experimental immunization with methylated bovine serum albumin-DNA complexes elicited antibodies to various forms of synthetic ssDNA/dsDNA, but notably not to mammalian dsDNA. Thus, for a long time with intense research, the general result was that all forms of ssDNA and dsDNA, but mammalian B helical DNA, had an immunogenic potential. Summarizing these results, a preliminary conclusion was settled, saying that mammalian dsDNA was not immunogenic while other forms of DNA were really immunogenic in the situation where they were in complex with proteins. Recent studies have focused on nuclease deficiencies as a condition where chromatin may be presented to the immune system in an immunogenic form. However, although such deficiencies may provide information as to how chromatin may be exposed and targeted by relevant antibodies, data demonstrate that nuclease deficiencies is not in general correlated with autoimmunity to components of chromatin. This review discusses these topics, and provides information that may explain processes that account for anti-dsDNA antibody responses in vivo.     

Rev1 promotes replication through UV lesions in conjunction with DNA polymerases η, ι, and κ but not DNA polymerase ζ

Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions; however, little is known about the protein factors that affect their function in human cells. In yeast, Rev1 plays a noncatalytic role as an indispensable component of Polζ, and Polζ together with Rev1 mediates a highly mutagenic mode of TLS. However, how Rev1 functions in TLS and mutagenesis in human cells has remained unclear. Here we determined the role of Rev1 in TLS opposite UV lesions in human and mouse fibroblasts and showed that Rev1 is indispensable for TLS mediated by Polη, Polι, and Polκ but is not required for TLS by Polζ. In contrast to its role in mutagenic TLS in yeast, Rev1 promotes predominantly error-free TLS opposite UV lesions in humans. The identification of Rev1 as an indispensable scaffolding component for Polη, Polι, and Polκ, which function in TLS in highly specialized ways opposite a diverse array of DNA lesions and act in a predominantly error-free manner, implicates a crucial role for Rev1 in the maintenance of genome stability in humans.     

POL32, a subunit of the Saccharomyces cerevisiae DNA polymerase δ, defines a link between DNA replication and the mutagenic bypass repair pathway

Pol32 is a subunit of Saccharomyces cerevisiae DNA polymerase δ required in DNA replication and repair. To gain insight into the function of Pol32 and to determine in which repair pathway POL32 may be involved, we extended the analysis of the pol32Δ mutant with respect to UV and methylation sensitivity, UV-induced mutagenesis; and we performed an epistasis analysis of UV sensitivity by combining the pol32Δ with mutations in several genes for postreplication repair (RAD6 group), nucleotide excision repair (RAD3 group) and recombinational repair (RAD52 group). These studies showed that pol32Δ is deficient in UV-induced mutagenesis and place POL32 in the error-prone RAD6/REV3 pathway. We also found that the increase in the CAN1 spontaneous forward mutation of different rad mutators relies entirely or partially on a functional POL32 gene. Moreover, in a two-hybrid screen, we observed that Pol32 interacts with Srs2, a DNA helicase required for DNA replication and mutagenesis. Simultaneous deletion of POL32 and SRS2 dramatically decreases cellular viability at 15 °C and greatly increases cellular sensitivity to hydroxyurea at the permissive temperature. Based on these findings, we propose that POL32 defines a link between the DNA polymerase and helicase activities, and plays a role in the mutagenic bypass repair pathway. Received: 25 May 2000 / Accepted: 3 July 2000     

Comparison of isoelectric focusing and polymerase chain reaction for the detection of β-lactamases

Extended spectrum β-lactamases (ESBLs) have been observed in virtually all the species of family Enterobacteriaceae. Threat posed by antibiotic resistance because of ESBLs is more serious as a number of technical problems are associated with the detection of these enzymes. Although a number of detection methods have been designed for ESBLs, every method has its own benefits and shortcomings as well. In earlier days, isoelectric focusing (IEF) was used as the gold standard for ESBL detection. This study was undertaken to compare IEF with polymerase chain reaction, a method which has been extensively used for ESBL detection these days.     

The suppression of the μ rhythm during the creation of imagery representation of movement

The aim of this study was to answer the following question: are there differences between the attenuation of μ rhythms, recorded with EEG in the parietal area during observation of movement and the creation of its imaginative representation? In addition, we checked the extent to which the μ rhythm suppression depends on whether the observed and the imagined movement is performed by a human or is artificial. As a result of the experiment a significant difference in μ rhythm suppression between the conditions “Observation,” “Imagery,” and “White noise” was recorded. It did not matter whether the motion was carried out by a human being or performed by a machine. The results are discussed in the light of findings which relate to the mirror neuron system.     

Isolation of new nonsense and frameshift mutants in the immunoglobulin μ heavy-chain gene of hybridoma cells

In order to expand the experimental material available for genetic and biochemical analyses of the natural immunoglobulin genes, we have isolated a variety of mutant mouse hybridoma cell lines. Some of these mutants have partial or complete deletions of the gene. Other mutants have nonsense or frameshift mutations in the exons encoding the variable and the second and third constant region domains of the heavy chain. When combined with earlier mutant data, this collection of genotypically and phenotypically tight mutants of known sequence spans most of the 10 kb of the gene, providing material for a variety of studies of genetic recombination and mRNA metabolism.     

c-mip down-regulates NF-κB activity and promotes apoptosis in podocytes

The mechanisms of podocyte disorders in cases of idiopathic nephrotic syndrome (INS) are complex and remain incompletely elucidated. The abnormal regulation of NF-κB may play a key role in the pathophysiology of these podocyte diseases, but at present, NF-κB has not been thoroughly investigated. In this study, we report that induction of c-mip in podocytes of patients with INS is associated with a down-regulation of RelA, a potent antiapoptotic factor that belongs to the NF-κB family. Overexpression of c-mip in differentiated podocytes promotes apoptosis by inducing caspase-3 activity and up-regulating the proapoptotic protein Bax, whereas the overall levels of the antiapoptotic protein Bcl-2 was concomitantly decreased. The associated overexpression of RelA prevented the proapoptotic effects of c-mip. In addition, the targeted induction of c-mip in podocytes in vivo inhibited the expression of the RelA protein and increased the Bax/Bcl-2 ratio. The expression of both c-mip and active caspase-3 increased in focal and segmental glomerulosclerosis biopsies, and both proteins displayed a close spatial relationship. These results suggest that alterations in NF-κB activity might result from the up-regulation of c-mip and are likely to contribute to podocyte disorders in cases of INS.     

Analysis of mutations introduced into the chromosomal immunoglobulin μ gene

We have introduced a pSV2neo-derived vector that contains a 2-base-pair (bp) deletion in its immunoglobulin gene constant region into hybridoma cells bearing a single copy of the wild-type chromosomal immunoglobulin gene. Homologous recombination between the transferred mutant C region and the wild-type chromosomal C region is expected to introduce the 2-bp deletion into the chromosomal gene, generating recombinant cells synthesizing noncytolytic IgM. Analysis of the DNA in independent noncytolytic transformants indicates that in one case the gene has the structure expected for correct homologous recombination. Unexpectedly, the remaining transformants, bear chromosomal gene deletions.