Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases.

Xeroderma pigmentosum is a monogenic disease characterized by hypersensitivity to ultraviolet light. The cells of xeroderma pigmentosum patients are defective in nucleotide excision repair, limiting their capacity to eliminate ultraviolet-induced DNA damage, and resulting in a strong predisposition to develop skin cancers. The use of rare cutting DNA endonucleases-such as homing endonucleases, also known as meganucleases-constitutes one possible strategy for repairing DNA lesions. Homing endonucleases have emerged as highly specific molecular scalpels that recognize and cleave DNA sites, promoting efficient homologous gene targeting through double-strand-break-induced homologous recombination. Here we describe two engineered heterodimeric derivatives of the homing endonuclease I-CreI, produced by a semi-rational approach. These two molecules-Amel3-Amel4 and Ini3-Ini4-cleave DNA from the human XPC gene (xeroderma pigmentosum group C), in vitro and in vivo. Crystal structures of the I-CreI variants complexed with intact and cleaved XPC target DNA suggest that the mechanism of DNA recognition and cleavage by the engineered homing endonucleases is similar to that of the wild-type I-CreI. Furthermore, these derivatives induced high levels of specific gene targeting in mammalian cells while displaying no obvious genotoxicity. Thus, homing endonucleases can be designed to recognize and cleave the DNA sequences of specific genes, opening up new possibilities for genome engineering and gene therapy in xeroderma pigmentosum patients whose illness can be treated ex vivo.

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.

Chromosome damage induced by combined treatments with restriction endonucleases introduced into CHO cells by single or double electroporation.

The possible recombination between non-homologous termini produced by restriction enzymes (REs) introduced in CHO cells by electroporation was studied. For this purpose, different combinations of REs that produced blunt or 5' overhanging DNA double-strand breaks were electroporated into cells either at the same time or separately by double electroporation experiments. Prior to double electroporation, it was confirmed that, once the cells have been electroporated, they resist a second electroporation, as assessed by cell viability analysis. Besides, the efficient and homogeneous introduction of labelled, non-permeable molecules was assessed by fluorescence microscopy. Our results showed interaction for most of the conditions, mainly when the REs were introduced separately. Differences found in the degree of interaction between the combinations studied are discussed.

Relationships between the cytotoxic effects of restriction endonucleases and radiation on mammalian cells.

Wild-type Chinese hamster ovary (CHO) cells, a radiosensitive mutant CHO line (xrs6), and two human cervical carcinoma cell lines, MS751 and ME180, differ in sensitivity to ionizing radiation with surviving fractions at 2 Gy (SF2) of 0.84, 0.06, 0.90 and 0.24 respectively. Restriction endonucleases (REs) were introduced into the cells by treatment with streptolysin O (SLO) and the effects of this on clonogenic cell survival were compared. A comparison was made of REs inducing either blunt- (AluI) or cohesive-ended (Sau3AI) double-strand breaks. Whilst MS751 cells were resistant to the effects of both REs, AluI caused significantly greater cell killing than Sau3AI in the other three lines (p < 0.05 for all). Both ME180 and xrs6 were significantly more sensitive to REs than their radioresistant counterparts, MS751 and CHO (p < 0.05 for both). In order to investigate the effect of DNA methylation on dsb induction, the isoschizomers MspI and HpaII (cohesive-ended dsb inducers) were introduced into the cell lines. Both REs recognize the same sequence but HpaII cannot cleave if the internal cytosine is methylated. MS751 was also resistant to the effects of both of these enzymes and MspI was more cytotoxic than HpaII in the other three lines (p < 0.03 for all). The differential sensitivity to the two REs was more marked in the radiosensitive cell lines, suggesting that there may be a greater degree of DNA methylation in radiosensitive cells. The variation in sensitivities to REs between the cell lines could not be explained in terms of differences in cell poration following SLO treatment because, although MS751 was resistant to SLO (25% of cells porated), the other three lines showed the same level of cell poration (98% of cells). With these four cell lines, there was a significant correlation between sensitivity to RE and radiosensitivity for AluI, Sau3AI and MspI but not for HpaII.

Molecular structural analysis of 417 HPRT mutations induced by restriction endonucleases in Chinese hamster ovary (CHO) cells.

CHO cells were exposed to 11 different restriction endonucleases by electroporation and their mutagenicity was measured. Nine of them have one or more recognition sites within exons of the HPRT gene, whereas the remaining two cut in introns only. The mutagenic efficiency of the various enzymes varied markedly; mutagenicity of Sau3AI was considerably higher than that of the other enzymes. Neither cytotoxicity nor mutagenicity could be related to the number or location of recognition sites within the cDNA. A total of 417 independent restriction enzyme induced mutant clones were isolated from 20 separate experiments for molecular analysis; all nine exons of the HPRT gene were analyzed by a modified multiplex deletion screening method with polymerase chain reaction (PCR) amplification. Among spontaneously arising mutants, 70.8% showed no change in PCR pattern, indicating a small scale change (point mutation), whereas partial deletions were observed in 24.7%, and total deletions in 4.5% of mutant clones. In contrast, approximately 70% of restriction enzyme induced mutants showed partial or total deletions. There was no obvious relationship between type of break (blunt versus staggered ends), and the DNA structure of the mutations induced. For partial deletions, the distribution of breakpoints within introns appeared to occur at random, and did not correlate with the mutagenicity of a given enzyme. Thus, though DNA double-strand breaks appear to be important mutagenic lesions that can induce a high frequency of deletion mutants, no specific relationship of mutagenic potential to the type of breaks, their sites within the HPRT gene or the molecular structure of the mutations induced could be identified.

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.

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.

Interaction between X-ray- and restriction endonuclease-induced lesions in the formation of chromosomal aberrations.

Experiments were performed to analyze the possible interaction between lesions induced by X-rays and restriction endonucleases in the production of chromosome-type exchanges. A stronger interaction was found between X-rays and the AluI-induced 'blunt termini' lesions than between X-rays and the BamHI-induced 'cohesive termini' lesions.

Restriction endonuclease Bam H I induces chromosomal aberrations in Chinese hamster ovary (CHO) cells.

Treatment of Chinese hamster ovary (CHO) cells with the restriction endonuclease Bam H I (recognition site: G/GATCC) leads to high frequencies of chromosomal aberrations. Experiments with bromodeoxyuridine-labelled chromosomes show that the aberrations occur nearly exclusively in first post-treatment metaphases. The results are interpreted to mean that only some of the cells take up the enzyme and that these cells are the ones showing the aberrations. Cells which do not take up the enzyme show up as differentially stained metaphases and have no aberrations. Why some cells take up the restriction enzyme and others not is not known, possibly this is dependent on the physiological condition of the cells.

Chromosomal aberrations induced by restriction endonucleases.

Restriction endonucleases (REs) are able to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells. The G1 phase of the cell cycle seems to be especially sensitive for the induction of chromosomal aberrations by REs. The different capacities of REs to induce chromosomal aberrations are probably correlated with the number of recognition sites in the genome.