The fate of linear DNA in Saccharomyces cerevisiae and Candida glabrata: the role of homologous and non-homologous end joining
Open accessAbstract
In vivo assembly of plasmids has become an increasingly used process, as high throughput studies in molecular biology seek to examine gene function. In this study, we investigated the plasmid construction technique called gap repair cloning (GRC) in two closely related species of yeast - Saccharomyces cerevisiae and Candida glabrata. GRC utilizes homologous recombination (HR) activity to join a linear vector and a linear piece of DNA that contains base pair homology. We demonstrate that a minimum of 20 bp of homology on each side of the linear DNA is required for GRC to occur with at least 10% efficiency. Between the two species, we determine that S. cerevisiae is slightly more efficient at performing GRC. GRC is less efficient in rad52 deletion mutants, which are defective in HR in both species. In dnl4 deletion mutants, which perform less non-homologous end joining (NHEJ), the frequency of GRC increases in C. glabrata, whereas GRC frequency only minimally increases in S. cerevisiae, suggesting that NHEJ is more prevalent in C. glabrata. Our studies allow for a model of the fate of linear DNA when transformed into yeast cells. This model is not the same for both species. Most significantly, during GRC, C. glabrata performs NHEJ activity at a detectable rate (>5%), while S. cerevisiae does not. Our model suggests that S. cerevisiae is more efficient at HR because NHEJ is less prevalent than in C. glabrata. This work demonstrates the determinants for GRC and that while C. glabrata has a lower efficiency of GRC, this species still provides a viable option for GRC.
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Genetic work involving generation of deletion/tagged strains in Candida glabrata has been hampered by its preference for non-homologous end joining (NHEJ) over homologous recombination, making gene targeting less efficient than in Saccharomyces cerevisiae.
“Furthermore, genetic work involving generation of deletion/tagged strains in C. glabrata has been hampered by its preference for non-homologous end joining (NHEJ) over homologous recombination, which is required for targeted integration of a DNA fragment at double-strand breaks (DSB), thus making gene targeting much less efficient than in S. cerevisiae (Ueno et al. 2007; Corrigan et al. 2013; Cen,”
From <i>Candida glabrata</i> : new tools and technologies—expanding the toolkit (2015) · cites this paper as Corrigan et al. 2013
NHEJ is more dominant over homologous recombination in C. glabrata.
“as NHEJ is more dominant over homologous recombination in C. glabrata (Ueno et al. 2007; Corrigan et al. 2013; Cen, Fiori and Dijck 2015)”
From <i>Candida glabrata</i> : new tools and technologies—expanding the toolkit (2015) · cites this paper as Corrigan et al. 2013
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Cites
Sikorski, R. S., & Hieter, P. (1989) · Genetics · doi:10.1093/genetics/122.1.19 · Cited by 8,685 · see citing works
Longtine, M. S., Mckenzie, A., DeMarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., & Pringle, J. R. (1998) · Yeast · doi:10.1002/(sici)1097-0061(199807)14:10 · Cited by 5,367 · see citing works
Mechanism of Eukaryotic Homologous Recombination
Filippo, J. S., Sung, P., & Klein, H. L. (2008) · Annual Review of Biochemistry · doi:10.1146/annurev.biochem.77.061306.125255 · Cited by 1,686 · see citing works
Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair
Symington, L. S. (2002) · Microbiology and Molecular Biology Reviews · doi:10.1128/mmbr.66.4.630-670.2002 · Cited by 965 · see citing works
Plasmid construction by homologous recombination in yeast
Ma, H., Kunes, S. M., Schatz, P. J., & Botstein, D. (1987) · Gene · doi:10.1016/0378-1119(87)90376-3 · Cited by 563 · see citing works
Recombination-mediated PCR-directed plasmid construction in vivo in yeast
Oldenburg, K. R. (1997) · Nucleic Acids Research · doi:10.1093/nar/25.2.451 · Cited by 520 · see citing works
Yeast DNA ligase IV mediates non-homologous DNA end joining
Wilson, T. E., Grawunder, U., & Lieber, M. R. (1997) · Nature · doi:10.1038/41365 · Cited by 418 · see citing works
Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae.
Schiestl, R. H., & Petes, T. D. (1991) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.88.17.7585 · Cited by 326 · see citing works
Hentges, P., Driessche, B. V., Tafforeau, L., Vandenhaute, J., & Carr, A. M. (2005) · Yeast · doi:10.1002/yea.1291 · Cited by 274 · see citing works
Teo, S., & Jackson, S. P. (1997) · The EMBO Journal · doi:10.1093/emboj/16.15.4788 · Cited by 259 · see citing works
Phosphate Transport and Sensing in Saccharomyces cerevisiae
Wykoff, D. D., & O’Shea, E. K. (2001) · Genetics · doi:10.1093/genetics/159.4.1491 · Cited by 238 · see citing works
Cormack, B. P., & Falkow, S. (1999) · Genetics · doi:10.1093/genetics/151.3.979 · Cited by 180 · see citing works
Rapid cloning by homologous recombinationin vivo
Bubeck, P., Winkler, M. E., & Bautsch, W. (1993) · Nucleic Acids Research · doi:10.1093/nar/21.15.3601 · Cited by 142 · see citing works
Minimum Length of Sequence Homology Required forin VivoCloning by Homologous Recombination in Yeast
Hua, S.-B., Qiu, M. S., Chan, E., Zhu, L., & Luo, Y. (1997) · Plasmid · doi:10.1006/plas.1997.1305 · Cited by 130 · see citing works
The Complete Set of Predicted Genes from Saccharomyces cerevisiae in a Readily Usable FormPreprint
Hudson, J. R., Dawson, E. P., Rushing, K. L., Jackson, C. H., Lockshon, D., Conover, D., Lanciault, C., Harris, J. R., Simmons, S. J., Rothstein, R. J., et al. (1997) · Genome Research · doi:10.1101/gr.7.12.1169 · Cited by 120 · see citing works
Tseng, H.-M., & Tomkinson, A. E. (2002) · Journal of Biological Chemistry · doi:10.1074/jbc.m206861200 · Cited by 111 · see citing works
Schiestl, R. H., Dominska, M., & Petes, T. D. (1993) · Molecular and Cellular Biology · doi:10.1128/mcb.13.5.2697-2705.1993 · Cited by 94 · see citing works
Isolation of a Candida glabrata centromere and its use in construction of plasmid vectors
Kitada, K., Yamaguchi, E., & Arisawa, M. (1996) · Gene · doi:10.1016/0378-1119(96)00132-1 · Cited by 70 · see citing works
Schiestl, R. H., Dominska, M., & Petes, T. D. (1993) · Molecular and Cellular Biology · doi:10.1128/mcb.13.5.2697 · Cited by 57 · see citing works
Ciudad, T., Andaluz, E., Steinberg‐Neifach, O., Lue, N. F., Gow, N. A. R., Calderone, R., & Larriba, G. (2004) · Molecular Microbiology · doi:10.1111/j.1365-2958.2004.04197.x · Cited by 56 · see citing works
The homologous recombination system of Ustilago maydis
Holloman, W. K., Schirawski, J., & Holliday, R. (2008) · Fungal Genetics and Biology · doi:10.1016/j.fgb.2008.04.006 · Cited by 52 · see citing works
Kitada, K., Yamaguchi, E., Hamada, K., & Arisawa, M. (1997) · Current Genetics · doi:10.1007/s002940050185 · Cited by 37 · see citing works
Compensatory role for Rad52 during recombinational repair in Ustilago maydis
Kojić, M., Mao, N., Zhou, Q., Lisby, M., & Holloman, W. K. (2008) · Molecular Microbiology · doi:10.1111/j.1365-2958.2008.06116.x · Cited by 34 · see citing works
Kerwin, C. L., & Wykoff, D. D. (2009) · Genetics · doi:10.1534/genetics.109.101063 · Cited by 34 · see citing works
Optimization of ordered plasmid assembly by gap repair in Saccharomyces cerevisiae
Eckert‐Boulet, N., Pedersen, M. L., Krogh, B. O., & Lisby, M. (2012) · Yeast · doi:10.1002/yea.2912 · Cited by 32 · see citing works
Plasmid Construction Using Recombination Activity in the Fission Yeast Schizosaccharomyces pombe
Chino, A., Watanabe, K., & Moriya, H. (2010) · PLoS ONE · doi:10.1371/journal.pone.0009652 · Cited by 26 · see citing works
Simple and Effective Gap-Repair Cloning Using Short Tracts of Flanking Homology in Fission Yeast
Matsuo, Y., Kishimoto, H., Horiuchi, T., Tanae, K., & Kawamukai, M. (2010) · Bioscience Biotechnology and Biochemistry · doi:10.1271/bbb.90967 · Cited by 19 · see citing works
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Research works printed but not linked: 1
- [18]Guthrie C, Fink GR (2002) Guide to yeast genetics and molecular and cell biology: Academic Press.
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- Authors
- Corrigan, M. W., Kerwin-Iosue, C. L., Kuczmarski, A. S., Amin, K. B., & Wykoff, D. D.
- Affiliations
- Villanova University (US)
- Corresponding author
- Wykoff, Dennis D.
- Venue
- PLoS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2013-07-24
- Volume
- 8
- Issue
- 7
- Pages
- e69628
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC3722132
- OpenAlex
- W2055369551
- Cited by
- 23
- References
- 27
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 2 · 2023: 2 · 2022: 3 · 2021: 2 · 2020: 3 · 2019: 3 · 2018: 1 · 2016: 2 · 2015: 3
- FWCI
- 0.75
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- PLoS ONE (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Figshare (submittedVersion, cc-by)
- Funders
- National Science Foundation (1121714)
- Villanova University
- National Institute of Environmental Health Sciences (T32ES007059)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Keywords
- homologous recombination, non-homologous end joining, Saccharomyces cerevisiae, Candida glabrata, linear DNA, plasmid assembly, RAD52, DNA repair
- MeSH terms
- DNA, Fungal, Recombination, Genetic, Saccharomyces cerevisiae, Candida glabrata, DNA End-Joining Repair
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