Purification and characterization of a novel ATP-independent type I DNA topoisomerase from a marine methylotroph

DNA topoisomerase is involved in DNA repair and replication. In this study, a novel ATP-independent 30-kDa type I DNA topoisomerase was purified and characterized from a marine methylotroph, Methylophaga sp. strain 3. The purified enzyme composed of a single polypeptide was active over a broad range of temperature and pH. The enzyme was able to relax only negatively supercoiled DNA. Mg(2+) was required for its relaxation activity, while ATP gave no effect. The enzyme was clearly inhibited by camptothecin, ethidium bromide, and single-stranded DNA, but not by nalidixic acid and etoposide. Interestingly, the purified enzyme showed Mn(2+)-activated endonuclease activity on supercoiled DNA. The N-terminal sequence of the purified enzyme showed no homology with those of other type I enzymes. These results suggest that the purified enzyme is an ATP-independent type I DNA topoisomerase that has, for the first time, been characterized from a marine methylotroph.     

4-Quinolones cause a selective loss of mitochondrial DNA from mouse L1210 leukemia cells

The 4-quinolone antibiotics nalidixic acid and ciprofloxacin and potent inhibitors of the bacterial type II topoisomerase DNA gyrase. Treatment of mouse L1210 leukemia cells with these drugs resulted in a delayed inhibition of cell proliferation. Prior to inhibition of cell proliferation, there was a time-dependent decrease in the cellular content of mitochondrial DNA (mtDNA). The decrease in mtDNA was associated with a decrease in the rate of mitochondrial respiration and an increase in the concentration of lactate in the growth medium. Inhibition of cell proliferation by 4-quinolones was reversible upon drug washout. However, there was a 2- to 4-day lag before the growth rate returned to normal levels. This was preceeded by an increase in mtDNA content and mitochondrial respiration. These studies suggest that inhibition of mammalian cell proliferation by 4-quinolone drugs is related to the selective depletion of mtDNA. © 1993 Wiley-Liss, Inc.     

Identification of DNA topoisomerases involved in immediate and transient DNA relaxation induced by heat shock inEscherichia coli

The linking number of plasmid DNA in exponentially growingEscherichia coli increases immediately and transiently after heat shock. The purpose of this study was to search for DNA topoisomerases that catalyze this relaxation of DNA. Neither introduction of atopA deletion mutation nor treatment of cells with DNA gyrase inhibitors affected the DNA relaxation induced by heat shock. Thus, DNA topoisomerase I and DNA gyrase are apparently not involved in the process. However, the reaction was inhibited by nalidixic acid or by oxolinic acid in thetopA mutant and the reaction was resistant to nalidixic acid in atopA mutant carrying, in addition, thenalA26 mutation. These results are interpreted as indicating that both DNA topoisomerase I and DNA gyrase are involved in the DNA relaxation induced by heat shock.     

Temperature shift-up leads to simultaneous and continuous plasmid DNA relaxation and induction of DnaK and GroEL proteins in anaerobically growing Escherichia coli cells

Abstract We previously reported that plasmid DNA in Escherichia coli cells growing under aerobic conditions relaxed immediately and transiently after heat shock (Mizushima, T., Natori, S. and Sekimizu, K., Mol. Gen. Genet. (1993) 238, 1–5). We next examined DNA relaxation and induction of heat shock proteins after heat shock in cells growing under anaerobic conditions. DNA in these cells relaxed rapidly (2 min) after heat shock (42°C), as was the case with aerobically growing cells, but full superhelicity was not recovered. The relaxed state of DNA topology was maintained for 60 min after heat shock. Induction of DnaK and GroEL proteins, which was transient in aerobically growing cells, was continuous in anaerobically growing cells. Therefore, induction of heat shock proteins correlated with DNA relaxation in both aerobic and anaerobic conditions.     

Induction of DNA ligase I by 1-beta-D-arabinosylcytosine and aphidicolin in MiaPaCa human pancreatic cancer cells

Exposure of MiaPaCa cells to 1-beta-D-arabinosylcytosine (ara-C) resulted in an increase in DNA ligase levels up to threefold compared to that in the untreated control cells, despite significant growth inhibition. Increased levels of DNA ligase I protein appear to correlate with the appearance of increased mRNA levels. The [(3)H]thymidine incorporation experiment and the biochemical assay of total polymerase activity revealed that an increase in DNA ligase I levels after treatment with ara-C was not accompanied by an increase of DNA synthesis or an increased presence of DNA polymerase activity inside cells. When cells resumed DNA synthesis after drug treatment, DNA ligase I levels began to drop, indicating that increased DNA ligase I is not required for DNA synthesis. An increase in DNA ligase I was also observed in cells treated with aphidicolin, another inhibitor of DNA synthesis that inhibits DNA polymerases without incorporating itself into DNA, indicating that an increase in DNA ligase I levels could be caused by the arrest of DNA replication by these agents. Interestingly, caffeine, which is a well-known inhibitor of DNA damage checkpoint kinases, abrogated the increase in DNA ligase I in MiaPaCa cells treated with ara-C and aphidicolin, suggesting that caffeine-sensitive kinases might be important mediators in the pathway leading to the increase in DNA ligase I levels in response to anticancer drugs, including ara-C and aphidicolin. We propose that ara-C and aphidicolin induce damage to the DNA strand by arresting DNA replication forks and subsequently increase DNA ligase I levels to facilitate repair of DNA damage.     

The mechanism of inhibition of DNA replication in HeLa S3 cells by the antitumor drug Ledakrin and other antitumor 1-nitro-9-aminoacridines

These studies were aimed at characterizing the capability of an antitumor DNA-damaging drug, Ledakrin, and its analogs to inhibit DNA replication in HeLa S3 cells. The studied agents are extremely potent inhibitors of [³H]thymidine incorporation in whole cells. These compounds produced also a potent dose- and time-dependent inhibition of DNA synthesis in subcellular systems derived from drug-treated cells, as found by [³H]dGTP incorporation in cellular lysates and nuclei. Experiments in which nuclei from control and drug-treated cells were supplemented with cytoplasmic fractions from either control or drug-treated cells, or with exogenous DNA, demonstrate that Ledakrin and other 1-nitro-9-aminoacridines inhibit DNA replication in HeLa S3 cells by interfering with the DNA template, while not affecting DNA polymerase(s) or other enzymes and replication factors. The negligible effect of Ledakrin added to lysates or nuclei from untreated cells suggests that metabolic activation is a prerequisite for replication inhibition by Ledakrin. Analysis of the size of newly synthesized DNA, by alkaline sucrose gradient sedimentation, indicates that Ledakrin does not inhibit the initiation of replication but does interfere with chain growth. Impairment of DNA replication by 1-nitro-9-aminoacridines seems to originate from DNA damage and to result in the inhibition of cellular growth.     

Co-induction of DNA relaxation and synthesis of DnaK and GroEL proteins inEscherichia coli by expression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor

We examined the influence of overexpression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor ofEscherichia coli, on DNA supercoiling and induction of heat shock proteins. Cells were transformed with a plasmid carrying the structural gene for LetD protein under control of thetac promoter, and LetD protein was induced by adding isopropyl-d-thiogalactopyranoside (IPTG) to the culture medium. Analysis by agarose gel electrophoresis in the presence of chloroquine revealed relaxation of plasmid DNA in cells depending on the concentration of IPTG employed for induction. Protein pulse-labeling experiments with [³⁵S]methionine and cysteine revealed that synthesis of DnaK and GroEL proteins was also induced by IPTG, and concentrations necessary for DNA relaxation and induction of the heat shock proteins were much the same. Expression of mutant LetD protein lacking two amino acid residues at the C-terminus induced neither DNA relaxation nor the synthesis of DnaK and GroEL proteins. Induction of wild-type LetD protein but not mutant LetD protein markedly enhanced synthesis of ³². We interpret these results to mean that DNA relaxation in cells caused by the expression of LetD protein induces heat shock proteins via increased synthesis of ³².     

Site-specific integration of retroviral DNA in human cells using fusion proteins consisting of human immunodeficiency virus type 1 integrase and the designed polydactyl zinc-finger protein E2C

During the life cycle of retroviruses, establishment of a productive infection requires stable joining of a DNA copy of the viral RNA genome into host cell chromosomes. Retroviruses are thus promising vectors for the efficient and stable delivery of genes in therapeutic protocols. Integration of retroviral DNA is catalyzed by the viral enzyme integrase (IN), and one salient feature of retroviral DNA integration is its lack of specificity, as many chromosomal sites can serve as targets for integration. Despite the promise for success in the clinic, one major drawback of the retrovirus-based vector is that any unintended insertion events from the therapy can potentially lead to deleterious effects in patients, as demonstrated by the development of malignancies in both animal and human studies. One approach to directing integration into predetermined DNA sites is fusing IN to a sequence-specific DNA-binding protein, which results in a bias of integration near the recognition site of the fusion partner. Encouraging results have been generated in vitro and in vivo using fusion protein constructs of human immunodeficiency virus type 1 IN and E2C, a designed polydactyl zinc-finger protein that specifically recognizes an 18-base pair DNA sequence. This review focuses on the method for preparing infectious virions containing the IN fusion proteins and on the quantitative PCR assays for determining integration site specificity. Efforts to engineer IN to recognize specific target DNA sequences within the genome may lead to development of effective retroviral vectors that can safely deliver gene-based therapeutics in a clinical setting.