Site-Specific Recombination Determined by I-Scei, a Mitochondrial Group I Intron-Encoded Endonuclease Expressed in the Yeast Nucleus

The Saccharomyces cerevisiae mitochondrial endonuclease I-SceI creates a double-strand break as the initiating step in the gene conversional transfer of the omega+ intron to omega- DNA. We have expressed a galactose-inducible synthetic I-SceI gene in the nucleus of yeast that also carries the I-SceI recognition site on a plasmid substrate. We find that the galactose-induced I-SceI protein can be active in the nucleus and efficiently catalyze recombination. With a target plasmid containing direct repeats of the Escherichia coli lacZ gene, one copy of which is interrupted by a 24-bp cutting site, galactose induction produces both deletions and gene conversions. Both the kinetics and the proportion of deletions and gene conversions are very similar to analogous events initiated by a galactose-inducible HO endonuclease gene. We also find that, in a rad52 mutant strain, the repair of double-strand breaks initiated by I-SceI and by HO are similarly affected: the formation of deletions is reduced, but not eliminated. Altogether, these results suggest either that the two endonucleases act in the same way after double-strand break formation or that the two endonucleases are not involved in subsequent steps.     

Interstitial deletions and intrachromosomal amplification initiated from a double-strand break targeted to a mammalian chromosome.

Interstitial deletions of tumour suppressor genes and amplification of oncogenes are two major manifestations of chromosomal instability in tumour cells. The development of model systems allowing the study of the events triggering these processes is of major clinical importance. Using the properties of the I-SceI nuclease to introduce a localized double-strand break (DSB) in a mammalian chromosome carrying its target sequence, we demonstrate here that both types of mutations can be initiated by non-conservative DSB repair pathways. In our system, I-SceI activity dissociates a transfected gpt gene from its promoter, allowing the isolation of gpt- clones. Our results show that intrachromatid single-strand annealing events occur frequently, giving rise to interstitial deletions not accompanied by other chromosomal rearrangements. We also observed that, when present in the cells, extrachromosomal DNA molecules are integrated preferentially at the broken locus. Taking advantage of the insertion of the I-SceI recognition sequence telomeric to and close to the dihydrofolate reductase gene, we show that a less frequent outcome of I-SceI activity is the initiation of cycles of intrachromosomal amplification of this marker, from breaks at a site merging with the enzyme target.     

Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination.

In mammalian cells, chromosomal double-strand breaks are efficiently repaired, yet little is known about the relative contributions of homologous recombination and illegitimate recombination in the repair process. In this study, we used a loss-of-function assay to assess the repair of double-strand breaks by homologous and illegitimate recombination. We have used a hamster cell line engineered by gene targeting to contain a tandem duplication of the native adenine phosphoribosyltransferase (APRT) gene with an I-SceI recognition site in the otherwise wild-type APRT+ copy of the gene. Site-specific double-strand breaks were induced by intracellular expression of I-SceI, a rare-cutting endonuclease from the yeast Saccharomyces cerevisiae. I-SceI cleavage stimulated homologous recombination about 100-fold; however, illegitimate recombination was stimulated more than 1,000-fold. These results suggest that illegitimate recombination is an important competing pathway with homologous recombination for chromosomal double-strand break repair in mammalian cells.     

Chromosomal aberrations induced by double strand DNA breaks

It has been suggested that introduction of double strand DNA breaks (DSBs) into mammalian chromosomes can lead to gross chromosomal rearrangements through improper DNA repair. To study this phenomenon, we employed a model system in which a double strand DNA break can be produced in human cells in vivo at a predetermined location. The ensuing chromosomal changes flanking the breakage site can then be cloned and characterized. In this system, the recognition site for the I-SceI endonuclease, whose 18 bp recognition sequence is not normally found in the human genome, is placed between a strong constitutive promoter and the Herpes simplex virus thymidine kinase (HSV-tk) gene, which serves as a negative selectable marker. We found that the most common mutation following aberrant DSB repair was an interstitial deletion; these deletions typically showed features of non-homologous end joining (NHEJ), such as microhomologies and insertions of direct or inverted repeat sequences. We also detected more complex rearrangements, including large insertions from adjacent or distant genomic regions. The insertion events that involved distant genomic regions typically represented transcribed sequences, and included both L1 LINE elements and sequences known to be involved in genomic rearrangements. This type of aberrant repair could potentially lead to gene inactivation via deletion of coding or regulatory sequences, or production of oncogenic fusion genes via insertion of coding sequences.     

The role of double-strand break-induced allelic homologous recombination in somatic plant cells

During meiosis, homologous recombination occurs between allelic sequences. To evaluate the biological significance of such a pathway in somatic cells, we used transgenic tobacco plants with a restriction site for the rare cutting endonuclease I-SceI within a negative selectable marker gene. These plants were crossed with two tobacco lines containing, in allelic position, either a deletion or an insertion within the marker gene that rendered both marker gene and restriction site inactive. After the double-strand break induction, we selected for repair events resulting in a loss of marker gene function. This loss was mostly due to deletions. We were also able to detect double strand break-induced allelic recombination in which the break was repaired by a faithful copying process from the homologue carrying the shortened transgene. The estimated frequency indicates that homologous recombination in somatic cells between allelic sites appears to occur at the same order of magnitude as between ectopic sites, and is thus far too infrequent to act as major repair pathway. As somatic changes can be transferred to the germ line, the prevalence of intrachromatid rearrangements over allelic recombination might be an indirect prerequisite for the enhanced genome plasticity postulated for plants.     

Capture of DNA sequences at double-strand breaks in mammalian chromosomes

To study double-strand break (DSB)-induced mutations in mammalian chromosomes, we transfected thymidine kinase (tk)-deficient mouse fibroblasts with a DNA substrate containing a recognition site for yeast endonuclease I-SceI embedded within a functional tk gene. To introduce a genomic DSB, cells were electroporated with a plasmid expressing endonuclease I-SceI, and clones that had lost tk function were selected. Among 253 clones analyzed, 78% displayed small deletions or insertions of several nucleotides at the DSB site. Surprisingly, approximately 8% of recovered mutations involved the capture of one or more DNA fragments. Among 21 clones that had captured DNA, 10 harbored a specific segment of the I-SceI expression plasmid mapping between two replication origins on the plasmid. Four clones had captured a long terminal repeat sequence from an intracisternal A particle (an endogenous retrovirus-like sequence) and one had captured what appears to be a cDNA copy of a moderately repetitive B2 sequence. Additional clones displayed segments of the tk gene and/or microsatellite sequences copied into the DSB. This first systematic study of DNA capture at DSBs in a mammalian genome suggests that DSB repair may play a considerable role in the evolution of eukaryotic genomes.     

Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells.

To analyze genomic changes resulting from double-strand break (DSB) repair, transgenic tobacco plants were obtained that carried in their genome a restriction site of the rare cutting endonuclease I-SceI within a negative selectable marker gene. After induction of DSB repair via Agrobacterium-mediated transient expression of I-SceI, plant cells were selected that carried a loss-of-function phenotype of the marker. Surprisingly, in addition to deletions, in a number of cases repair was associated with the insertion of unique and repetitive genomic sequences into the break. Thus, DSB repair offers a mechanism for spreading different kinds of sequences into new chromosomal positions. This may have evolutionary consequences particularly for plants, as genomic alterations occurring in meristem cells can be transferred to the next generation. Moreover, transfer DNA (T-DNA), carrying the open reading frame of I-SceI, was found in several cases to be integrated into the transgenic I-SceI site. This indicates that DSB repair also represents a pathway for the integration of T-DNA into the plant genome.     

Subtelomeric regions in mammalian cells are deficient in DNA double-strand break repair

We have previously demonstrated that double-strand breaks (DSBs) in regions near telomeres are much more likely to result in large deletions, gross chromosome rearrangements, and chromosome instability than DSBs at interstitial sites within chromosomes. In the present study, we investigated whether this response of subtelomeric regions to DSBs is a result of a deficiency in DSB repair by comparing the frequency of homologous recombination repair (HRR) and nonhomologous end joining (NHEJ) at interstitial and telomeric sites following the introduction of DSBs by I-SceI endonuclease. We also monitored the frequency of small deletions, which have been shown to be the most common mutation at I-SceI-induced DSBs at interstitial sites. We observed no difference in the frequency of small deletions or HRR at interstitial and subtelomeric DSBs. However, the frequency of NHEJ was significantly lower at DSBs near telomeres compared to interstitial sites. The frequency of NHEJ was also lower at DSBs occurring at interstitial sites containing telomeric repeat sequences. We propose that regions near telomeres are deficient in classical NHEJ as a result of the presence of cis-acting telomere-binding proteins that cause DSBs to be processed as though they were telomeres, resulting in excessive resection, telomere loss, and eventual chromosome rearrangements by alternative NHEJ.     

Single molecule PCR reveals similar patterns of non-homologous DSB repair in tobacco and Arabidopsis

DNA double strand breaks (DSBs) occur constantly in eukaryotes. These potentially lethal DNA lesions are repaired efficiently by two major DSB repair pathways: homologous recombination and non-homologous end joining (NHEJ). We investigated NHEJ in Arabidopsis thaliana and tobacco (Nicotiana tabacum) by introducing DNA double-strand breaks through inducible expression of I-SceI, followed by amplification of individual repair junction sequences by single-molecule PCR. Using this process over 300 NHEJ repair junctions were analysed in each species. In contrast to previously published variation in DSB repair between Arabidopsis and tobacco, the two species displayed similar DSB repair profiles in our experiments. The majority of repair events resulted in no loss of sequence and small (1-20 bp) deletions occurred at a minority (25-45%) of repair junctions. Approximately ~1.5% of the observed repair events contained larger deletions (>20 bp) and a similar percentage contained insertions. Strikingly, insertion events in tobacco were associated with large genomic deletions at the site of the DSB that resulted in increased micro-homology at the sequence junctions suggesting the involvement of a non-classical NHEJ repair pathway. The generation of DSBs through inducible expression of I-SceI, in combination with single molecule PCR, provides an effective and efficient method for analysis of individual repair junctions and will prove a useful tool in the analysis of NHEJ.     

Non-homologous end-joining for repairing I-SceI-induced DNA double strand breaks in human cells

DNA double strand breaks (DSBs) are usually repaired through either non-homologous end-joining (NHEJ) or homologous recombination (HR). While HR is basically error-free repair, NHEJ is a mutagenic pathway that leads to deletion. NHEJ must be precisely regulated to maintain genomic integrity. To clarify the role of NHEJ, we investigated the genetic consequences of NHEJ repair of DSBs in human cells. Human lymphoblastoid cell lines TSCE5 and TSCE105 have, respectively, single and double I-SceI endonuclease sites in the endogenous thymidine kinase gene (TK) located on chromosome 17q. I-SceI expression generated DSBs at the TK gene. We used the novel transfection system (Amaxa Nucleofector) to introduce an I-SceI expression vector into the cells and randomly isolated clones. We found mutations involved in the DSBs in the TK gene in 3% of TSCE5 cells and 30% of TSCE105 cell clones. Most of the mutations in TSCE5 were small (1-30bp) deletions with a 0-4bp microhomology at the junction. The others consisted of large (>60) bp deletions, an insertion, and a rearrangement. Mutants resulting from interallelic HR also occurred, but infrequently. Most of the mutations in TSCE105, on the other hand, were deletions that encompassed the two I-SceI sites generated by NHEJ at DSBs. The sequence joint was similar to that found in TSCE5 mutants. Interestingly, some mutants formed a new I-SceI site by perfectly joining the two original I-SceI sites without deletion of the broken-ends. These results support the idea that NHEJ for repairing I-SceI-induced DSBs mainly results in small or no deletions. Thus, NHEJ must help maintain genomic integrity in mammalian cells by repairing DSBs as well as by preventing many deleterious alterations.     

Deletions at short direct repeats and base substitutions are characteristic mutations for bleomycin-induced double- and single-strand breaks, respectively, in a human shuttle vector system.

Using the radiomimetic drug, bleomycin, we have determined the mutagenic potential of DNA strand breaks in the shuttle vector pZ189 in human fibroblasts. The bleomycin treatment conditions used produce strand breaks with 3'-phosphoglycolate termini as > 95% of the detectable dose-dependent lesions. Breaks with this end group represent 50% of the strand break damage produced by ionizing radiation. We report that such strand breaks are mutagenic lesions. The type of mutation produced is largely determined by the type of strand break on the plasmid (i.e. single versus double). Mutagenesis studies with purified DNA forms showed that nicked plasmids (i.e. those containing single-strand breaks) predominantly produce base substitutions, the majority of which are multiples, which presumably originate from error-prone polymerase activity at strand break sites. In contrast, repair of linear plasmids (i.e. those containing double-strand breaks) mainly results in deletions at short direct repeat sequences, indicating the involvement of illegitimate recombination. The data characterize the nature of mutations produced by single- and double-strand breaks in human cells, and suggests that deletions at direct repeats may be a 'signature' mutation for the processing of DNA double-strand breaks.     

Recombination events during integration of transfected DNA into normal human cells.

The mechanisms of recombination responsible for random integration of transfected DNA into the genome of normal human cells have been investigated by analysis of plasmid-cell DNA junctions. Cell clones containing integrated plasmid sequences were selected by morphological transformation of primary human fibroblasts after transfection with a plasmid containing simian virus 40 sequences. Nucleotide sequence analysis of the plasmid-cell DNA junctions was performed on cloned DNA fragments containing the integration sites from two of these cell clones. Polymerase chain reaction was then performed with human cell DNA from primary fibroblasts to isolate the cell DNA from the same sites before plasmid integration. Comparison of the sequences at the plasmid-cell DNA junctions with those of both the original plasmid and the cell DNA demonstrated short sequence similarities and additional nucleotides, typical of nonhomologous recombination. Evidence of short deletions in the cell DNA at the plasmid integration sites suggests that integration occurred by a mechanism similar to that used for repair of spontaneous or gamma ray-induced strand breaks. Plasmid integration occurred within nonrepetitive cell DNA with no major rearrangements, although rearrangements of the cell DNA at the integration site occurred in one of the clones after integration.     

Site-specific integration of Agrobacterium tumefaciens T-DNA via double-stranded intermediates

Agrobacterium tumefaciens-mediated genetic transformation involves transfer of a single-stranded T-DNA molecule (T strand) into the host cell, followed by its integration into the plant genome. The molecular mechanism of T-DNA integration, the culmination point of the entire transformation process, remains largely obscure. Here, we studied the roles of double-stranded breaks (DSBs) and double-stranded T-DNA intermediates in the integration process. We produced transgenic tobacco (Nicotiana tabacum) plants carrying an I-SceI endonuclease recognition site that, upon cleavage with I-SceI, generates DSB. Then, we retransformed these plants with two A. tumefaciens strains: one that allows transient expression of I-SceI to induce DSB and the other that carries a T-DNA with the I-SceI site and an integration selection marker. Integration of this latter T-DNA as full-length and I-SceI-digested molecules into the DSB site was analyzed in the resulting plants. Of 620 transgenic plants, 16 plants integrated T-DNA into DSB at their I-SceI sites; because DSB induces DNA repair, these results suggest that the invading T-DNA molecules target to the DNA repair sites for integration. Furthermore, of these 16 plants, seven plants incorporated T-DNA digested with I-SceI, which cleaves only double-stranded DNA. Thus, T-strand molecules can be converted into double-stranded intermediates before their integration into the DSB sites within the host cell genome.     

Differential Requirement for SUB1 in Chromosomal and Plasmid Double-Strand DNA Break Repair

Non homologous end joining (NHEJ) is an important process that repairs double strand DNA breaks (DSBs) in eukaryotic cells. Cells defective in NHEJ are unable to join chromosomal breaks. Two different NHEJ assays are typically used to determine the efficiency of NHEJ. One requires NHEJ of linearized plasmid DNA transformed into the test organism; the other requires NHEJ of a single chromosomal break induced either by HO endonuclease or the I-SceI restriction enzyme. These two assays are generally considered equivalent and rely on the same set of NHEJ genes. PC4 is an abundant DNA binding protein that has been suggested to stimulate NHEJ. Here we tested the role of PC4''s yeast homolog SUB1 in repair of DNA double strand breaks using different assays. We found SUB1 is required for NHEJ repair of DSBs in plasmid DNA, but not in chromosomal DNA. Our results suggest that these two assays, while similar are not equivalent and that repair of plasmid DNA requires additional factor(s) that are not required for NHEJ repair of chromosomal double-strand DNA breaks. Possible roles for Sub1 proteins in NHEJ of plasmid DNA are discussed.     

Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae.

The mitochondrial intron-encoded endonuclease I-SceI of Saccharomyces cerevisiae has an 18-bp recognition sequence and, therefore, has a very low probability of cutting DNA, even within large genomes. We demonstrate that double-strand breaks can be initiated by the I-SceI endonuclease at a predetermined location in the mouse genome and that the breaks can be repaired with a donor molecule homologous regions flanking the breaks. This induced homologous recombination is approximately 2 orders of magnitude more frequent than spontaneous homologous recombination and at least 10 times more frequent than random integration near an active promoter. As a consequence of induced homologous recombination, a heterologous novel sequence can be inserted at the site of the break. This recombination can occur at a variety of chromosomal targets in differentiated and multipotential cells. These results demonstrate homologous recombination involving chromosomal DNA by the double-strand break repair mechanism in mammals and show the usefulness of very rare cutter endonucleases, such as I-SceI, for designing genome rearrangements.     

A role for DNA mismatch repair protein Msh2 in error-prone double-strand-break repair in mammalian chromosomes

We examined error-prone nonhomologous end joining (NHEJ) in Msh2-deficient and wild-type Chinese hamster ovary cell lines. A DNA substrate containing a thymidine kinase (tk) gene fused to a neomycin-resistance (neo) gene was stably integrated into cells. The fusion gene was rendered nonfunctional due to a 22-bp oligonucleotide insertion, which included the 18-bp I-SceI endonuclease recognition site, within the tk portion of the fusion gene. A double-strand break (DSB) was induced by transiently expressing the I-SceI endonuclease, and deletions or insertions that restored the tk-neo fusion gene's reading frame were recovered by selecting for G418-resistant colonies. Overall, neither the frequency of recovery of G418-resistant colonies nor the sizes of NHEJ-associated deletions were substantially different for the mutant vs. wild-type cell lines. However, we did observe greater usage of terminal microhomology among NHEJ events recovered from wild-type cells as compared to Msh2 mutants. Our results suggest that Msh2 influences error-prone NHEJ repair at the step of pairing of terminal DNA tails. We also report the recovery from both wild-type and Msh2-deficient cells of an unusual class of NHEJ events associated with multiple deletion intervals, and we discuss a possible mechanism for the generation of these "discontinuous deletions."     

Suppression of high-fidelity double-strand break repair in mammalian chromosomes by pifithrin-alpha,a chemical inhibitor of p53

We investigated the effect of pifithrin-alpha (PFTalpha), a chemical inhibitor of p53, on DNA double-strand break (DSB) repair in mammalian chromosomes. Thymidine kinase-deficient mouse fibroblasts were stably transfected with DNA substrates containing one or two recognition sites for yeast endonuclease I-SceI embedded within a herpes simplex virus thymidine kinase gene. Genomic DSBs were induced by introducing an I-SceI expression plasmid into cells in the presence or absence of 20 microM PFTalpha. From cells containing the DNA substrate with a single I-SceI site we recovered low-fidelity nonhomologous end-joining (NHEJ) events in which one or more nucleotides were deleted or inserted at the DSB. From cells containing the substrate with two I-SceI sites we recovered high-fidelity DNA end-joining (precise ligation (PL)) events. We found that treatment of cells with PFTalpha caused a 5-10-fold decrease in recovery of PL but decreased recovery of NHEJ by less than two-fold. Deletion sizes associated with NHEJ were unaffected by treatment with PFTalpha. Our work suggests the possibility that p53 facilitates high-fidelity DSB repair while playing little or no role in mutagenic NHEJ.     

Two unlinked double-strand breaks can induce reciprocal exchanges in plant genomes via homologous recombination and nonhomologous end joining

Using the rare-cutting endonuclease I-SceI we were able to demonstrate before that the repair of a single double-strand break (DSB) in a plant genome can be mutagenic due to insertions and deletions. However, during replication or due to irradiation several breaks might be induced simultaneously. To analyze the mutagenic potential of such a situation we established an experimental system in tobacco harboring two unlinked transgenes, each carrying an I-SceI site. After transient expression of I-SceI a kanamycin-resistance marker could be restored by joining two previously unlinked broken ends, either by homologous recombination (HR) or by nonhomologous end joining (NHEJ). Indeed, we were able to recover HR and NHEJ events with similar frequencies. Despite the fact that no selection was applied for joining the two other ends, the respective linkage could be detected in most cases tested, demonstrating that the respective exchanges were reciprocal. The frequencies obtained indicate that DSB-induced translocation is up to two orders of magnitude more frequent in somatic cells than ectopic gene conversion. Thus, DSB-induced reciprocal exchanges might play a significant role in plant genome evolution. The technique applied in this study may also be useful for the controlled exchange of unlinked sequences in plant genomes.     

Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease.

To maintain genomic integrity, double-strand breaks (DSBs) in chromosomal DNA must be repaired. In mammalian systems, the analysis of the repair of chromosomal DSBs has been limited by the inability to introduce well-defined DSBs in genomic DNA. In this study, we created specific DSBs in mouse chromosomes for the first time, using an expression system for a rare-cutting endonuclease, I-SceI. A genetic assay has been devised to monitor the repair of DSBs, whereby cleavage sites for I-SceI have been integrated into the mouse genome in two tandem neomycin phosphotransferase genes. We find that cleavage of the I-SceI sites is very efficient, with at least 12% of stably transfected cells having at least one cleavage event and, of these, more than 70% have undergone cleavage at both I-SceI sites. Cleavage of both sites in a fraction of clones deletes 3.8 kb of intervening chromosomal sequences. We find that the DSBs are repaired by both homologous and nonhomologous mechanisms. Nonhomologous repair events frequently result in small deletions after rejoining of the two DNA ends. Some of these appear to occur by simple blunt-ended ligation, whereas several others may occur through annealing of short regions of terminal homology. The DSBs are apparently recombinogenic, stimulating gene targeting of a homologous fragment by more than 2 orders of magnitude. Whereas gene-targeted clones are nearly undetectable without endonuclease expression, they represent approximately 10% of cells transfected with the I-SceI expression vector. Gene targeted clones are of two major types, those that occur by two-sided homologous recombination with the homologous fragment and those that occur by one-sided homologous recombination. Our results are expected to impact a number of areas in the study of mammalian genome dynamics, including the analysis of the repair of DSBs and homologous recombination and, potentially, molecular genetic analyses of mammalian genomes.