Inhibition of DNA replication of human papillomavirus by using zinc finger-single-chain FokI dimer hybrid.

Previously, we reported that an artificial zinc-finger protein (AZP)-staphylococcal nuclease (SNase) hybrid (designated AZP-SNase) inhibited DNA replication of human papillomavirus type 18 (HPV-18) in mammalian cells by binding to and cleaving a specific HPV-18 ori plasmid. Although the AZP-SNase did not show any side effects under the experimental conditions, the SNase is potentially able to cleave RNA as well as DNA. In the present study, to make AZP hybrid nucleases that cleave only viral DNA, we switched the SNase moiety in the AZP-SNase to the single-chain FokI dimer (scFokI) that we had developed previously. We demonstrated that transfection with a plasmid expressing the resulting hybrid nuclease (designated AZP-scFokI) inhibited HPV-18 DNA replication in transient replication assays using mammalian cells more efficiently than AZP-SNase. Then, by linker-mediated PCR analysis, we confirmed that AZP-scFokI cleaved an HPV-18 ori plasmid around its binding site in mammalian cells. Finally, a modified MTT assay revealed that AZP-scFokI did not show any significant cytotoxicity. Thus, the newly developed AZP-scFokI hybrid is expected to serve as a novel antiviral reagent for the neutralization of human DNA viruses with less fewer potential side effects.

Creating designed zinc-finger nucleases with minimal cytotoxicity.

Zinc-finger nucleases (ZFNs) have emerged as powerful tools for delivering a targeted genomic double-strand break (DSB) to either stimulate local homologous recombination with investigator-provided donor DNA or induce gene mutations at the site of cleavage in the absence of a donor by nonhomologous end joining both in plant cells and in mammalian cells, including human cells. ZFNs are formed by fusing zinc-finger proteins to the nonspecific cleavage domain of the FokI restriction enzyme. ZFN-mediated gene targeting yields high gene modification efficiencies (>10%) in a variety of cells and cell types by delivering a recombinogenic DSB to the targeted chromosomal locus, using two designed ZFNs. The mechanism of DSB by ZFNs requires (1) two ZFN monomers to bind to their adjacent cognate sites on DNA and (2) the FokI nuclease domains to dimerize to form the active catalytic center for the induction of the DSB. In the case of ZFNs fused to wild-type FokI cleavage domains, homodimers may also form; this could limit the efficacy and safety of ZFNs by inducing off-target cleavage. In this article, we report further refinements to obligate heterodimer variants of the FokI cleavage domain for the creation of custom ZFNs with minimal cellular toxicity. The efficacy and efficiency of the reengineered obligate heterodimer variants of the FokI cleavage domain were tested using the green fluorescent protein gene targeting reporter system. The three-finger and four-finger zinc-finger protein fusions to the REL_DKK pair among the newly generated FokI nuclease domain variants appear to eliminate or greatly reduce the toxicity of designer ZFNs to human cells.

SCE formation after exposure of CHO cells prelabelled with BrdU or biotin-dUTP to various DNA-damaging agents.

Formation of sister chromatid exchanges (SCE) is a mechanism of repair or bypass of DNA damage during S phase. Although SCE have been studied for a long time, the types of DNA lesions involved and the role of 5-bromodeoxyuridine (BrdU) in SCE formation are a matter of debate. We have developed a novel method of differential labelling of sister chromatids with biotin-16-2'-deoxyuridine-5'-triphosphate (biotin-dUTP) and could show that a substantial proportion of radiation-induced SCE arise via damage to BrdU-moieties. The present investigations were performed to examine the role of BrdU in the formation of SCE by the endonucleases AluI and DNase I, as well as the alkylating agent mitomycin C (MMC). CHO cells unifilarily prelabelled with biotin-dUTP or BrdU were treated and the frequencies of SCE analysed in the first post-treatment mitoses. AluI induced similar frequencies of SCE in cells prelabelled with BrdU or biotin-dUTP. DNase I induced significantly more SCE in cells prelabelled with BrdU than with biotin-dUTP. MMC induced slightly more SCE in cells labelled with biotin-dUTP than BrdU, but the difference was not significant. The possible mechanisms responsible for the enhanced SCE frequency following DNase I treatment of cells prelabelled with BrdU are discussed.

HaeIII induces position-dependent chromosomal breakage in barley (Hordeum vulgare L.).

The pattern of localized chromosomal breakage induced by the restriction endonuclease HaeIII in reconstructed barley karyotypes T-1586 and T-21 was investigated. It was found that nucleolus organizing regions (NORs) of chromosomes 6 and 7 (segments 46 and 38, respectively), containing actively transcribed ribosomal (r)DNA, as well as segments 39 and 47, both containing condensed rDNA repeats, are the most pronounced aberration hot-spots in T-1586. The number of aberrations observed in these segments was three to five times higher than theoretically expected. The intrachromosomal distribution of chromatid aberrations in karyotype T-21, where the NOR-bearing segments in chromosomes 6 and 7 change their position, revealed a substantial difference in the aberration hot-spot behaviour. A position-specific increase in aberration clustering was observed, most pronounced in segments 38 and 47. On the other hand, segment 46 retained its initial sensitivity, while segment 39 in the new position lost its previous status as a mutation hot-spot. The data are indicative of the expressivity of aberration hot-spots generated after treatment with this restriction endonuclease being influenced by their distinct chromosomal location.

Ultrasound permeabilizes CHO cells for the endonucleases AluI and benzon nuclease.

Ultrasound permeabilizes Chinese hamster ovary (CHO) cells for the endonucleases AluI and benzon nuclease which leads to the induction of chromosomal aberrations by these enzymes. A few aberrant cells were observed when trypsinized cells or adherent cells were exposed to the enzymes in the absence of ultrasound. Our data show that sonication can be used to introduce endonucleases into CHO cells. We further demonstrate that few cells can internalize endonucleases without previous permeabilization.

Restriction endonucleases induce chromosomal aberrations in barley.

The clastogenic ability of the restriction endonucleases (MspI, HpaII and HaeIII) in germinating seeds of reconstructed barley karyotype was assessed. An effective induction of chromosomal aberrations after restrictase treatment was observed. The frequency, types and cell-cycle dependence of the observed abnormalities are discussed in relation to the distinct characteristics of the enzymes and the features of the plant genome. The capacity to induce aberrations was not significantly influenced by the nature of the double-strand breaks (blunt- or cohesive-ended); however, it was dependent on the methylation status of the plant DNA. The restriction enzymes displayed an S-independent mode of action revealing the transition between G1 and S as the most sensitive stage of the cell cycle in barley for induction of chromosomal damage.

Chromosomal aberrations induced in human whole blood cultures by pipetting cell pellets in the presence of AluI.

A physical method is described to permeabilize human peripheral lymphocytes in culture for the restriction endonuclease AluI. Blood cultures are incubated for 20 h, pelleted, strongly pipetted with different types of Pasteur pipettes in the presence of AluI and recovered for up to 54 h in the presence of 5-bromodeoxyuridine. Up to 20% of aberrant first posttreatment (M1) metaphases with chromosome-type aberrations are found. The method works with whole blood cultures and is therefore easy to perform.

Exposure of CHO cells to AluI: comparison of chromosomal aberrations and cell survival.

Chinese hamster ovary (CHO) cells were exposed to various doses of the restriction endonuclease AluI in the presence of 2.2 M glycerol. Some of the cells were cultured for analysis of chromosomal aberrations and some for analysis of colony-forming ability. Cell killing is mainly mediated by chromosomal aberrations.

Modulatory effect of sodium butyrate on AluI-induced chromosomal aberrations in CHO cells.

Exponentially growing CHO cells exposed to millimolar concentrations of sodium butyrate (SB) for 24 h were treated with AluI using two methods of cell poration, i.e., electroporation and streptolysin O (SLO). Under both conditions, SB was found to induce a 2-4-fold increase in AluI-induced chromosomal aberrations. When cells in monolayer were treated with AluI/SLO, lower concentrations of SB (2.5 mM) and AluI (1-4 U/ml) were required to produce a similar effect as that observed for electroporated cells, demonstrating the differential sensitivity of the two methods. Furthermore, in AluI/SLO-treated cells, a higher percentage of cells was found to show increased frequencies of aberrations per cell, compared to AluI/electroporated cells. The mechanism by which SB modulates the cell response to AluI treatment might involve changes in chromatin configuration thereby increasing the accessibility of AluI to different parts of chromatin.

Combination treatments of Chinese hamster ovary cells with various restriction endonucleases result in chromosomal aberrations whose frequencies are additive or less than additive.

Chinese hamster ovary cells were treated with combinations of different restriction endonucleases (RE). The frequencies of chromosomal aberrations after combination treatments were additive or less than additive when compared with the effects of the single RE. These data indicate that DNA double-strand breaks (DSB) induced by different types of RE in combination treatments lead to chromosomal aberrations in the same way as DSB induced by single RE.

Chromatid-type aberrations induced by AluI in Chinese hamster ovary cells.

Treatment of CHO cells with AluI in the S-phase leads to chromatid-type aberrations whose frequencies are linearly correlated with dose. Treatment in the S-phase leads to fewer aberrations than treatment in the G1-phase, which is comparable to chromosomal aberration induction following X-irradiation in the G1- and S-phases. Treatment in the G1-phase leads to few chromatid-type interchanges, some of these may originate from DNA single-strand gaps induced by AluI in canonical structures of DNA and in DNA.RNA hybrids.

Mutagenic effects of restriction enzymes in Chinese hamster cells: evidence for high mutagenicity of Sau3AI at the hprt locus.

CHO cells were exposed to seven different restriction endonucleases by electroporation and their cytotoxicity and mutagenicity measured. Cell killing as determined by a colony formation assay occurred in a concentration-dependent manner for each enzyme. The D0 of the survival curves were: MspI = 24U; AluI = 31U; Sau3AI = 106U; HaeIII = 46U; HinfI = 30U; PvuII = 35U; BamHI = 163U. BamHI and Sau3AI were particularly ineffective in cell killing. For the 6-base recognition sequence enzymes, PvuII (a blunt-ended cutter) was much more cytotoxic per unit electroporated than BamHI (a sticky-ended cutter). Among the 4-base cutters, Sau3AI and HaeIII were generally less cytotoxic than HinfI or PvuII. Cell killing appeared to depend on the nature of the recognition sequence and cutting sites rather than on the cutting frequency. The mutagenic effects of these restriction endonucleases were investigated by measuring the induced frequencies of hprt gene mutations. The mutagenicity of Sau3AI was dramatically higher than the other enzymes, increasing linearly with dose up to 35U. When normalized for survival, the mutagenicity of Sau3AI relative to the other enzymes was even greater. The mutagenic effect of BamHI, which has the same 5' protruding site as Sau3AI, was much lower at similar dose and survival levels. MspI, BamHI, and PvuII which have no recognition sites within the hprt coding sequence were marginally- or non-mutagenic. Based on these results and the distribution of cutting sites within the hprt cDNA for the enzymes studied, the hypothesis is discussed that a region in exon 4 is highly sensitive to the induction of mutants by DNA double-strand breaks.