Current Studies of Biotechnology Volume II. - Environment PLASMID INTEGRATION IN YEAST: CONCEPTIONS AND MISSCONCEPTIONS ZORAN ZGAGA, KREŠIMIR GJURAČIĆ 1, IVAN-KREŠIMIR SVETEC, PETAR T. MITRIKESKI AND SANDRA GREGORIĆ Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia; *PLIVA Research institute,zagreb, Croatia Abstract Integration of exogenous DNA into the yeast genome occurs by homologous recombination and is both directed and stimulated by the presence of double-strand breaks (DSBs) in transforming DNA. Repair of the break with or without plasmid integration occurs with equal probability and it is generally assumed that no information is lost or gained during this process. However, some important assumptions of current models are challenged by the results obtained by our group. First, we showed that rare illegitimate integrations may also occur in the presence of homology in the yeast genome and that integration in homology does not always result in reconstruction of the functional allele. Second, the presence of point mutations in double-stranded, but not in single-stranded plasmid, decreased integration in homology. However this antirecombinogenic effect was not proportional to the number of mutations. Third, targeted integrations were strongly suppressed by the presence of short heterologous sequences at the ends of the linearized plasmid. Terminal heterology is not always eliminated during integration and this process can lead to the generation of new alleles. In our experimental system, less then 10% of transformants that repaired DSB present on the replicative plasmid contained the plasmid sequence in their chromosomal DNA. We propose that the plasmid integration can be stimulated by repair processes operating on the hybrid DNA formed during recombination. Key words: gene targeting, genetic recombination, Saccharomyces cerevisiae Introduction Application of different technologies for gene transfer and/or modification is often limited by recombinational machinery of the host cell. Illegitimate integration of exogenous DNA is associated with alterations in the organization of genetic material that may lead to unpredictable changes in cell physiology and genetic stability. Unfortunately, this seems to be the prevailing mechanism of genetic transformation in a number of organisms, including human cells. Theoretically, this problem could be circumvented either by stimulation of homologous recombination, or by suppression of illegitimate integration and much effort has been made in order to better understand these processes. For the study of homologous recombination, the yeast Saccharomyces cerevisiae is still the model organism of choice. Although illegitimate events have also been described and characterised in yeast (1, 2, 3) homologous integration occurs with high frequency and is routinely used for manipulation of yeast genes. In first transformation experiments circular, non-replicative plasmids were found to recombine with homology present in the genomegiving rise to plasmid integration or to genetic conversion of the mutated allele (4). Later it was shown that restriction enzyme-induced double-strand breaks (DSBs) on the plasmid molecule stimulate transformation and direct recombination process to the homology present in the yeast genome (5). These experiments were the basis for development of the DSB model for genetic recombination (6), but also presented the first example of gene targeting technology. Subsequently, another procedure based on homologous recombination was developed, making possible gene replacement/disruption in a single step (7). By different experimental strategies yeast genes can now be mutated, deleted, replaced or cloned and foreign DNA can be introduced into any desired location within the genome. Corresponding author address: Zoran Zgaga, Faculty of Food Technology and Biotechnology, Pierotti St. 6, 10000 Zagreb, Croatia
ZORAN ZGAGA ET AL 136 Several models have been proposed to explain different pathways of genetic recombination in yeast (8). Plasmid integration is taken as an example of conservative reciprocal recombination where no new sequences are gained or lost. After transformation with linearized plasmid molecule recombination is initiated in homology by 3' single-stranded ends and is followed by strand exchange leading to the formation of hybrid DNA. Resolution of the crossed structures (Holliday junctions) will either separate recombining molecules or integrate the whole plasmid into homology (Fig 1). Recombination between plasmids and yeast chromosomes occurs by similar mechanism and may reflect recombinational repair of spontaneous lesions. In order to get deeper insight in molecular mechanism of this process we investigated the process of plasmid integration. Obtained results challenge some important assumptions of the current models for homologous recombination. Fig 1. DSB-induced homologous recombination between plasmid molecule and yeast chromosome. Hybrid DNA is formed by strand exchange in the region of homology (A). Two crossed structures (Holliday junctions) may be resolved in either direction giving rise to plasmid integration (C) or DSB repair without integration (B). Material and Methods Yeast transformation, Southern blot analysis, strains and integrative plasmids pcw12, pab218-5 and pom have been described previously (9, 10, 11). The replicative plasmids prom and psg2 (Figure 4) contain yeast origin of replication (ARS1) and are constructed by conventional procedures (12). Results and Discussion ILLEGITIMATE VS. HOMOLOGOUS PLASMID INTEGRATION First demonstration of illegitimate recombination in yeast was made in transformation experiments with plasmid molecules that shared no extended homology with genomic DNA (1). Plasmids were linearized with different restriction enzymes before transformation and among rare transformants three types of recombination events could be distinguished: (a) insertion at sites of very short terminal homology, (b) insertion next to sequences recognized by topoisomerase I and (c) ligation to the fragments of the yeast mitochondrial DNA (2). Interestingly, if restriction enzyme used to cut plasmid molecule was not extracted before transformation, plasmid sequence could also be found integrated in recognition sequences present in the yeast genome. Subsequent studies elucidated genetic requirements for illegitimate recombination in yeast (13). The frequency of illegitimate integration was very low, typically less then one transformant per μg of plasmid DNA (1, 3). We wondered whether illegitimate integrations also occur in the presence of homology, but are undetected due to their low frequency. Experimental system designed in Figure 2 was developed in order to answer this question. For transformation experiments we used the integrative plasmid pcw12 (3), containing the yeast URA3 and ARG4 genes, that was cut within the ARG4 gene with restriction endonuclease BsmI. The recipient strain was auxotrophic for uracil and arginine since the URA3 gene contained the Ty element inserted within the coding region (ura3-52 allele) while the 3' end of the ARG4 gene was deleted (Figure 2A). Transformants selected as uracil prototrophs were further replica-plated on the arginine omission medium. For Ura +, Arg + transformants it was assumed that plasmid integration occurred by homologous recombination with the chromosomal arg4 allele. Only Ura +, Arg - transformants were further analysed by Southern blotting in order to detect possible illegitimate events. The results of this analysis are presented in Figure 2B and they clearly demonstrate that illegitimate integrations may occur even in the presence of homology, but are about thousand-
PLASMID INTEGRATION IN YEAST: CONCEPTIONS AND MISSCONCEPTIONS 137 fold less frequent than integration into homology. Interestingly, three independent Ura +, Arg - transformants contained plasmid molecule integrated in the arg4 gene. Mutations in the ARG4 gene could occur as a consequence of imprecise recombination or error-prone DNA synthesis during DSB-repair as was already reported for the gap repair process in yeast (14). However, we can not rule out the possibility that mutations in the ARG4 gene occurred before transformation, during plasmid propagation in the E. coli cells. Fig 2. Illegitimate integrations in the presence of homology. A. Experimental system. Arg +, Ura + transformants can be produced only by targeted plasmid integration to the arg4 locus. B. Results. Genetic and molecular analysis revealed four classes of transformants: 1. Arg +, Ura + transformants produced by targeted integration, 2. Arg -, Ura + transformants produced by targeted integration, 3. Arg -, Ura + transformants produced by conversion of the Ty element present in the ura3-52 allele and 4. Arg -, Ura + transformants produced by illegitimate integration event. Asterisk denotes deletion in the promoter region of the ARG4 gene. A hundredfold increase in the frequency of illegitimate integration coupled with gross genomic rearrangements was observed after yeast transformation with single-stranded DNA (3). Although the mechanism of this process is still obscure, it could have contributed in shaping of the actual yeast genome structure (15). Therefore, the addition of denaturated "carrier" DNA suggested in some transformation protocols (16) should be avoided, since it may induce undesirable changes in the genome of the host strain. HETEROLOGOUS ENDS IN GENE TARGETING The ends of a broken DNA molecule are potent initiators of homologous recombination. We wondered whether the presence of a short terminal heterology will influence this process. Integrative plasmid pab218-5 was constructed with a 102 bp heterologous insertion within the CYC1 gene. Insertion was created by head-tohead ligation of two polylinker sequences (17). Before transformation plasmids were cut within the insert with restriction enzymes leaving heterologous ends of variable length (Figure 3A). We observed a size-dependent inhibition of plasmid integration in the presence of heterologous sequences at the ends of the broken molecule (17, 18). For heterologies longer than 30 bp we typically obtained less than 10% of transformants compared to the plasmid with no heterology present at the ends. Transformants obtained with plasmids containing 51 bp of terminal heterology were further analysed by Southern blotting. Only 3/100 transformants analysed contained the plasmid sequence integrated somewhere outside the CYC1 region while all other transformants were produced by targeted integration. Interestingly, 19/97 transformants targeted to the CYC1 region included at least several bases present in the terminal heterology (Figure 3B). In another set of experiments a plasmid with different heterologous ends was used and we detected 4/100 transformants that included short heterologous sequence in the target homology (data not shown). These results may be summarized as follows: short terminal heterologies decrease the frequency of targeted integrations, but are not always eliminated as predicted by current models for homologous recombination in yeast (8). The proportion of illegitimate integrations (3/100) observed in these experiments was higher then in experiments presented in Figure 2 (2/3203). However, the overall efficiency of transformation was decreased
ZORAN ZGAGA ET AL 138 for 90-95% due to the decrease in targeted integrations. Therefore, the yield of illegitimate transformants observed in these two assays is comparable. In other words, suppression of targeted integration did not result in significant increase in illegitimate integration, suggesting that these two processes are not in competition. Opposite conclusion was reached by Gherbi et al. (19) who found that the inactivation of the homologue of the yeast gene RAD50 resulted in increased intrachromosomal homologous recombination in the plant Arabidopsis thaliana. This difference may reflect different control of homologous vs. illegitimate recombination in these two organisms or may be due to different recombination assays used in these studies. Fig 3. The role of heterologous ends in plasmid integration. A. Experimental system. DSBs induced in the insertion present in the plasmid CYC1 gene leave identical heterologous ends from 0 to 51 bp (17). B. Transformation events revealed by Southern blot analysis: 1. targeted plasmid integration with elimination of heterologous ends, 2. targeted plasmid integration without complete elimination of heterologous ends, 3. conversion of the Ty element present in the ura3-52 allele and 4. illegitimate integration. SUBSTRATE REQUIREMENTS FOR INTEGRATION OF CIRCULAR PLASMIDS Efficiency of recombination between DNA molecules involved in homologous recombination is influenced by their size and sequence polymorphism. For integration of circular, non-replicative plasmid into yeast chromosome the question of substrate requirements was addressed in the study of Koren et al (10). The socalled "minimal efficient processing segment" (MEPS) needed for recombination was mapped between 321 and 363 bp, in accordance with the values observed in other experimental systems. However, rare integrations were also observed with shorter fragments (107 and 259 bp) indicating the existence of a MEPS-independent process for homologous recombination. Point mutations decreased integration to homology, but the inhibitory effect of these mutations was not cumulative. These results indicate that the influence of homology size and polymorphism on homologous recombination between plasmid molecule and yeast chromosome could be more complex then previously reported. Interestingly, integration of the single-stranded (ss) plasmid was not affected by the low sequence polymorphism analysed in this study (10). This result suggests that the plasmids isolated in the ss-form could be used for introduction of polymorphic sequences into the yeast genome. DSB REPAIR-ASSOCIATED PLASMID INTEGRATION Aberrant genetic segregations in meiosis are often associated with reciprocal recombination of the flanking markers. In mitotic cells, repair of the DSB or gap present on the replicative plasmid was first shown to be closely associated with reciprocal recombination so that about 50% of transformants contained plasmid molecule integrated in the genome (5). Later studies, using different experimental systems, indicated that this could rather be an exception then a rule, since only about 10% of recombination events in mitotic cells resulted in reciprocal recombination (8). Since this point is of major interest for gene manipulations we decided to
PLASMID INTEGRATION IN YEAST: CONCEPTIONS AND MISSCONCEPTIONS 139 reconsider the relationship between DSB repair and plasmid integration in yeast. We prepared replicative plasmids that could be targeted to different regions of the yeast genome with restriction enzyme-induced DSBs (Figure 4). Two classes of transformants were scored: stable transformants, that contained the plasmid molecule integrated in their genome and transformants that were unstable since they contained reporter gene on the plasmid molecule. Therefore, the proportion of stable transformants indicated the proportion of repair events that ended with reciprocal recombination. In order to prevent the repair of DSBs by simple ligation of the sticky ends created by restriction enzymes we deleted the LIG4 gene (20) in the host strain. Fig 4. DSB repair-associated plasmid integration. The plasmid prom contained a DSB (A) or 389 bp gap (B) in the CYC1 region. The plasmid psg2 was targeted to the URA3 gene with restriction endonuclease NcoI (C) and the same plasmid preparation was also used for transformation after filling of recessed 3' ends with the Klenow fragment (D). For repair events targeted to the CYC1 region (858 bp) by a DSB induced with KpnI, less then 5% of transformants contained plasmid molecule integrated in the genome (Figure 4A). Similar value was observed with the plasmid that contained a 389 bp gap in the CYC1 gene (Figure 4B) and with the plasmid that contained six point mutations in the CYC1 region (not shown). In another set of experiments repair events were targeted to the ura3-52 locus. This allele contains a 6,1 kb Ty1 insertion and repair events were directed downstream from the insertion by a DSB created with restriction endonuclease NcoI. A slight increase in the proportion of stable transformants was observed, but this difference was not statistically significant. However, when the ends of the broken molecule were filled in with the Klenow fragment, an increase in the proportion of stable transformants was observed (P=0,01). It should be noted that in this case the Ura + transformants could arise only if the "surplus" of homology at the end of the plasmid molecule is eliminated before or during recombination process. All these results demonstrate important departure in the proportion of integration events from expected 50% predicted by the DSB model for homologous recombination (6) and the repair process could rather proceed through "synthesis-dependent strand annealing" (SDSA) pathway (8). Different results were obtained in another
ZORAN ZGAGA ET AL 140 study, where only those recombination events were selected that resulted in the loss of the heterologous insertion from the ura3-52 allele (21). Among 76 transformants analysed, 29 contained plasmid molecule integrated in the genome (38,1%), which is close to the expected 50%. Therefore, it seems that the repair of a DSB present on the plasmid molecule may proceed by either pathway. Those repair events that involve formation of the heteroduplex DNA and gene conversion or degradation of redundant homology may have greater chance to be resolved as cross-overs. Concluding remarks For more than twenty years integration of exogenous DNA into the yeast genome is used as a model for targeted manipulations of the eukaryotic genome. General concepts developed in yeast gave important contribution to the progress made in this area on model organisms like mustard weed, fruitfly and mice. Recent results, including the ones presented here, show that this process is more complex then indicated by initial studies and may lead to transformation-associated genetic alterations described in other organisms. Future studies will concentrate on genetic control and molecular mechanisms of different transformation-associated recombination processes, but also on speciffic issues like, for example, mechanisms of DNA uptake and nuclear localization or homology search process. References 1. Schiestel RH, Petes TD. Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 1991; 88:7585-7589. 2. Schiestel RH, Dominska M, Petes TD. Transformation of Saccharomyces cerevisiae with nonhomologous DNA: illegitimate integration of transforming DNA into yeast chromosomes and in vivo ligation of transforming DNA to mitochondrial DNA sequences. Mol Cell Biol 1993; 13:2697-2705. 3. Gjuračić K, Zgaga Z. Illegitimate integration of single-stranded DNA in Saccharomyces cerevisiae. Mol Gen Genet 1996; 253:173-181. 4. Hinnen A, Hicks JB, Fink GR. Transformation of yeast. Proc Natl Acad Sci USA 1978;75:1929-1933. 5. Orr-Weaver TL, Szostak JW. Yeast recombination: the association between double-strand gap repair and crossing-over. Proc Natal Acad Sci USA 1983; 80:4417-4421. 6. Szostak JW, Orr-Weaver TL, Rothstein RJ, Stahl FW. The double-strand-break repair model for recombination. Cell 1983; 33:25-35. 7. Rothstein RJ. One-step gene disruption in yeast. Methods enzymol 1983; 101:202-211. 8. Paques F, Haber JE. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbial Mol Biol Rev 1999; 63:349-404. 9. Zgaga Z, Chanet R, Radman M, Fabre F. Mismatch-stimulated plasmid integration in yeast. Curr Genet 1991;19:329-332. 10. Koren P, Svetec IK, Mitrikeski PT, Zgaga Z. The influence of homology size and polymorphism on plasmid integration in the yeast CYC1 region. Curr Genet 2000; 37:292-297. 11. Svetec IK, Stjepandić D, Borić V, Mitrikeski PT, Zgaga Z. The influence of a palindromic insertion on plasmid integration in yeast. Food technol biotechnol 2001; 39:169-173. 12. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2 nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1989. 13. Schiestel RH, Zhu J, Petes TD. Effects of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae. Mol Cell Biol 1994; 14:4493-4500. 14. Strathern JN, Shafer BK, McGill CB. DNA synthesis errors associated with double-strand-break repair. Genetics 1995; 140:965-972. 15. Wolfe KH, Shields DC. Molecular evidence for an ancient duplication of the entire yeast genome. Nature 1997; 387:708-713. 16. Schiestl RH, Gietz RD. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet 1989; 16:339-346. 17. Zgaga Z. Repair of plasmid DNA in the yeast Saccharomyces cerevisiae. Ph. D. Thesis, University of Zagreb 1990. 18. Svetec IK. The influence of short heterologous sequences on plasmid integration in the yeast Saccharomyces cerevisiae genome. Master of Science Thesis, University of Zagreb 2000. 19. Gherbi H, Gallego MG, Jalut N, Lucht JM, Hohn B, White CI. Homologous recombination in plants is stimulated in the absence of Rad50. EMBO reports 2001; 2:287-291. 20. Teo SH, Jackson SP. Identification of Saccharomyces cerevisiae DNA ligase IV: involvement in DNA double-strand break repair. EMBO J 1997; 16:4788-4795. 21. Mitrikeski PT. Homologous recombination between plasmid and yeast gene interrupted by heterologous insertion. Master of Science Thesis, University of Zagreb 2001.