Akio Matsukage

FATTY ACIDS SELECTIVELY INHIBIT EUKARYOTIC DNA POLYMERASE ACTIVITIES IN VITRO

The in vitro relationship between eukaryotic DNA polymerases and fatty acids was investigated. Some fatty acids strongly inhibited the activities of DNA polymerase alpha and/or beta in vitro. The kinetics of inhibition by linoleic acid showed that DNA polymerase alpha was non-competitively inhibited with respect to the DNA template and substrate (dTTP), while DNA polymerase beta was inhibited competitively with both DNA and substrate.

The inhibitory action of fatty acids on DNA polymerase β

We found previously that long-chain fatty acids could inhibit eukaryotic DNA polymerase activities in vitro [1,2]. The purpose of the present study was to investigate the mode of this inhibition in greater detail. Among the C18 to C24 fatty acids examined, the strongest inhibitor was a C24 fatty acid, nervonic acid (NA), and the weakest was a C18 fatty acid, linoleic acid (LA). We analyzed the inhibitory effect of these two fatty acids and their modes of action. For DNA polymerase beta (pol. beta), NA acted by competing with both the substrate- and template-primer, but for DNA polymerase alpha (pol. alpha) or human immunodeficiency virus type 1 reverse transcriptase (HIV-1 reverse transcriptase or HIV-RT), NA acted non-competitively. NA-binding to pol. beta could be stopped with a non-ionic detergent, but the binding to pol. alpha or HIV-RT could not. The inhibition mode of LA showed the same characteristics, except that the minimum inhibitory dose of the longer chain was much lower. We also tested the effects of NA and LA using pol. beta and its proteolytic fragments, as described by Kumar et al. [3,4]. Both of the fatty acids were found to bind to the 8 kDa DNA-binding domain fragment, and to suppress binding to the template-primer DNA. We found that 10,000 times more of either fatty acid was required for it to bind to the 31 kDa catalytic domain or inhibit the DNA polymerase activity. The possible modes of inhibition by these long-chain fatty acids are discussed, based on the present findings.

Identification of ter94 , Drosophila VCP , as a modulator of polyglutamine-induced neurodegeneration

We have successfully generated a Drosophila model of human polyglutamine (polyQ) diseases by the targeted expression of expanded-polyQ (ex-polyQ) in the Drosophila compound eye. The resulting eye degeneration is progressive and ex-polyQ dosage- and ex-polyQ length-dependent. Furthermore, intergenerational changes in repeat length were observed in homozygotes, with concomitant changes in the levels of degeneration. Through genetic screening, using this fly model, we identified loss-of-function mutants of the ter94 gene that encodes the Drosophila homolog of VCP/CDC48, a member of the AAA+ class of the ATPase protein family, as dominant suppressors. The suppressive effects of the ter94 mutants on ex-polyQ-induced neurodegeneration correlated well with the degrees of loss-of-function, but appeared not to result from the inhibition of ex-polyQ aggregate formation. In the ex-polyQ-expressing cells of the late pupa, an upregulation of ter94 expression was observed prior to cell death. Co-expression of ter94 with ex-polyQ severely enhanced eye degeneration. Interestingly, when ter94 was overexpressed in the eye by increasing the transgene copies, severe eye degeneration was induced. Furthermore, genetical studies revealed that ter94 was not involved in grim-, reaper-, hid-, ced4-, or p53-induced cell death pathways. From these observations, we propose that VCP is a novel cell death effector molecule in ex-polyQ-induced neurodegeneration, where the amount of VCP is critical. Control of VCP expression may thus be a potential therapeutic target in ex-polyQ-induced neurodegeneration.

Difference in the expression level of DNA polymerase β among mouse tissues: High expression in the pachytene spermatocyte

The expression level of DNA polymerase beta was determined in various mouse tissues. Northern blot hybridization analysis using rat cDNA as a probe revealed that the mRNA of about 1.5 kb for this enzyme is present in all kinds of tissues examined, but its content widely varies among tissues; the most abundant DNA polymerase beta mRNA was present in the testis, which was followed by brain, thymus, and spleen. The mRNA content was low in heart, kidney, and liver. In testis and brain, two minor species of transcripts of 3.3 and 6.2 kb were detected in addition to that of 1.5 kb. DNA polymerase beta activities in these tissues were closely correlated with the mRNA content, indicating that the expression of this enzyme is mainly regulated by the level of the mRNA. A survey of DNA polymerase beta mRNA levels in the testes at successive postnatal developmental stages and in isolated spermatogenic cells indicated that DNA polymerase beta mRNA was most abundant in spermatocytes at early pachytene. Since meiotic recombination occurs in this period, DNA polymerase beta may be involved in the repair-type DNA synthesis associated with the recombination process.

Inhibition of DNA polymerase α, DNA polymerase β, terminal deoxynucleotidyl transferase, and DNA ligase II by poly(ADP-ribosyl)ation reaction in vitro☆

Incubation of DNA polymerase α1, DNA polymerase β, terminal deoxynucleotidyl transferase, or DNA ligase II in a reconstituted poly(ADP-ribosyl)ating enzyme system markedly suppressed the activity of these enzymes. Components required for poly(ADP-ribose) synthesis including poly(ADP-ribose) polymerase, NAD+, DNA, and Mg2+ were all essential for the observed suppression. Purified poly(ADP-ribose) itself, however, was slightly inhibitory to all of these enzymes. Furtheremore, the suppressed activities of DNA polymerase α, DNA polymerase β, and terminal deoxynucleotidyl transferase were largely restored (3 to 4-fold stimulation was observed) by a mild alkaline treatment, a procedure known to hydrolyze alkaline-labile ester linkage between poly(ADP-ribose) and an acceptor protein. All of these results strongly suggest that the four nuclear enzymes were inhibited as a result of poly(ADP-ribosyl)ation of either the enzyme molecule itself or some regulatory proteins of these enzymes.

Ectopic Expression of DREF Induces DNA Synthesis, Apoptosis, and Unusual Morphogenesis in the Drosophila Eye Imaginal Disc: Possible Interaction with Polycomb and trithorax Group Proteins

ABSTRACT The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory element DRE (5′-TATCGATA) in addition to E2F recognition sites. A specific DRE-binding factor, DREF, positively regulates DRE-containing genes. In addition, it has been reported that DREF can bind to a sequence in the hsp70 scs′ chromatin boundary element that is also recognized by boundary element-associated factor, and thus DREF may participate in regulating insulator activity. To examine DREF function in vivo, we established transgenic flies in which ectopic expression of DREF was targeted to the eye imaginal discs. Adult flies expressing DREF exhibited a severe rough eye phenotype. Expression of DREF induced ectopic DNA synthesis in the cells behind the morphogenetic furrow, which are normally postmitotic, and abolished photoreceptor specifications of R1, R6, and R7. Furthermore, DREF expression caused apoptosis in the imaginal disc cells in the region where commitment to R1/R6 cells takes place, suggesting that failure of differentiation of R1/R6 photoreceptor cells might cause apoptosis. The DREF-induced rough eye phenotype was suppressed by a half-dose reduction of the E2F gene, one of the genes regulated by DREF, indicating that the DREF overexpression phenotype is useful to screen for modifiers of DREF activity. Among Polycomb/trithorax group genes, we found that a half-dose reduction of some of the trithorax group genes involved in determining chromatin structure or chromatin remodeling (brahma, moira, and osa) significantly suppressed and that reduction of Distal-lessenhanced the DREF-induced rough eye phenotype. The results suggest a possibility that DREF activity might be regulated by protein complexes that play a role in modulating chromatin structure. Genetic crosses of transgenic flies expressing DREF to a collection ofDrosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the DREF-induced rough eye phenotype. These deletions should be useful to identify novel targets of DREF and its positive or negative regulators.

Studies on inhibitors of mammalian DNA polymerase α and β: Sulfolipids from a pteridophyte, Athyrium niponicum

Abstract Three sulfolipid compounds, 1, 2 and 3 , have been isolated from a higher plant, a pteridophyte, Athyrium niponicum , as potent inhibitors of the activities of calf DNA polymerase α and rat DNA polymerase β. The inhibition by the sulfolipids was concentration dependent, and almost complete inhibition of DNA polymerase α and DNA polymerase β was achieved at 6 and 8 μg/mL, respectively. The compounds did not influence the activities of calf thymus terminal deoxynucleotidyl transferase, prokaryotic DNA polymerases such as the Klenow fragment of DNA polymerase I, T4 DNA polymerase and Taq polymerase, the DNA metabolic enzyme DNase I, and even a DNA polymerase from a higher plant, cauliflower. Similarly, the compounds did not inhibit the activity of the human immunodeficiency virus type 1 reverse transcriptase. The kinetic studies of the compounds showed that DNA polymerase α was inhibited non-competitively with respect to the DNA template and substrate, whereas DNA polymerase β was inhibited competitively with both the DNA template and substrate. The binding to DNA polymerase β could be stopped with non-ionic detergent, but the binding to DNA polymerase α could not.

Polyester wax embedding and sectioning technique for immunohistochemistry.

We have developed a method useful for immunohistochemical studies by combining tissue fixation with buffered neutral formalin and polyester wax embedding. Buffered neutral formalin fixation preserves cell and tissue fine structure, and also the antigenicity of unstable enzymes. Polyester wax embedding makes possible thin serial sections of various tissues and preserves antigenicities for at least 6 months. We have demonstrated using this technique the localization of alpha-amylase in mouse salivary gland, parietal-cell specific antigen in mouse glandular stomach, and DNA polymerase alpha and beta in chick tissue.

Striking similarity of the distribution patterns of the poly(ADP-ribose) polymerase and DNA polymerase β among various mouse organs

The expression level of poly(ADP-ribose) polymerase mRNA as well as the level of enzymatic activity were examined in various mouse organs by northern blot and activity gel analyses. High levels of the mRNA expression and enzymatic activity were observed in testis, thymus, spleen, and brain. On the other hand, low levels of the mRNA expression and enzymatic activity were observed in liver and kidney. These findings suggest that the expression of the poly(ADP-ribose) polymerase is mainly regulated by transcription. Striking similarity was observed between the patterns of organ distribution of enzymatic activities of poly(ADP-ribose) polymerase and DNA polymerase beta in various mouse organs.

Roles of multiple promoter elements of the proliferating cell nuclear antigen gene during Drosophila development

The expression of genes involved in DNA replication is closely correlated with the proliferating state of cells and is repressed with the progression of differentiation during development. Promoter regions of the Drosophila proliferating cell nuclear antigen (PCNA) gene and the DNA polymerase α gene contain a common 8‐base pair promoter element (DRE: DNA replication‐related element). The examination of a common expression mechanism for DNA replication‐related genes, which is regulated positively by growth signals and negatively by differentiation signals would be of interest.  We generated PCNA‐LacZ fusion genes in which the 5′‐flanking sequence of the PCNA gene has been mutated. An examination of the expression of these fusion genes, introduced into flies by germ‐line transformation, led to the identification of another distinct regulatory element, URE (upstream regulatory element), within the region from –168 to –119 with respect to the transcription initiation site. During embryogenesis, the region containing the DRE sequence (–108 to –91) greatly stimulated the PCNA gene minimal promoter (–86 to +130), when it was placed upstream of the promoter in both normal and reverse orientations. Addition of the URE sequence further stimulated the promoter activity twofold. During larval stages, both DRE and URE were indispensable to the promoter activity, since neither of the sequences alone activated the minimal promoter. Demonstration of β‐galactosidase activity indicated URE plays an essential role in various larval tissues such as salivary gland and imaginal disc. While the minimal promoter region alone directed maternal expression of lacZ in ovaries of adult females, both DRE and URE further stimulated promoter activity.  These results show several elements of the PCNA gene promoter play roles during Drosophila development.