Ty1 integrase overexpression leads to integration of non-Ty1 DNA fragments into the genome of Saccharomyces cerevisiae

Friedl, Anna A.; Kiechle, Markus; Maxeiner, Horst G.; Schiestl, Robert H.; Eckardt‐Schupp, Friederike

doi:10.1007/s00438-010-0561-4

Cited by 10 publications

(10 citation statements)

References 48 publications

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“…In vivo , IN overexpression from the GAL1 promoter allows the integration of blunt double-stranded DNA fragments, which have no similarity with Ty1 DNA, except for the conserved terminal dinucleotides (5′-TG-CA-3′) [176]. Most insertion events present hallmarks of IN-mediated events, such as 5-bp TSDs and Ty1 target site preferences.…”

Section: Post-translational Steps In Retrotranspositionmentioning

confidence: 99%

The Ty1 LTR-Retrotransposon of Budding Yeast,Saccharomyces cerevisiae

2015

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Summary Long-terminal repeat (LTR)-retrotransposons generate a copy of their DNA (cDNA) by reverse transcription of their RNA genome in cytoplasmic nucleocapsids. They are widespread in the eukaryotic kingdom and are the evolutionary progenitors of retroviruses [1]. The Ty1 element of the budding yeast Saccharomyces cerevisiae was the first LTR-retrotransposon demonstrated to mobilize through an RNA intermediate, and not surprisingly, is the best studied. The depth of our knowledge of Ty1 biology stems not only from the predominance of active Ty1 elements in the S. cerevisiae genome but also the ease and breadth of genomic, biochemical and cell biology approaches available to study cellular processes in yeast. This review describes the basic structure of Ty1 and its gene products, the replication cycle, the rapidly expanding compendium of host co-factors known to influence retrotransposition and the nature of Ty1's elaborate symbiosis with its host. Our goal is to illuminate the value of Ty1 as a paradigm to explore the biology of LTR-retrotransposons in multicellular organisms, where the low frequency of retrotransposition events presents a formidable barrier to investigations of retrotransposon biology.

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Section: Post-translational Steps In Retrotranspositionmentioning

confidence: 99%

The Ty1 LTR-Retrotransposon of Budding Yeast,Saccharomyces cerevisiae

2015

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“…2(A)). Ty integrase can also facilitate the integration of non-Ty1 DNA fragments similar to cDNA containing the conserved terminal dinucleotide 5′-TG-CA-3′ [18]. …”

Section: Rna On the Frontlines Of Dna Damagementioning

confidence: 99%

DNA repair by RNA: Templated, or not templated, that is the question

2016

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Cells are continuously exposed to both endogenous and exogenous sources of genomic stress. To maintain chromosome stability, a variety of mechanisms have evolved to cope with the multitude of genetic abnormalities that can arise over the life of a cell. Still, failures to repair these lesions are the driving force of cancers and other degenerative disorders. DNA double-strand breaks (DSBs) are among the most toxic genetic lesions, inhibiting cell ability to replicate, and are sites of mutations and chromosomal rearrangements. DSB repair is known to proceed via two major mechanisms: homologous recombination (HR) and non-homologous end joining (NHEJ). HR reliance on the exchange of genetic information between two identical or nearly identical DNA molecules offers increased accuracy. While the preferred substrate for HR in mitotic cells is the sister chromatid, this is limited to the S and G2 phases of the cell cycle. However, abundant amounts of homologous genetic substrate may exist throughout the cell cycle in the form of RNA. Considered an uncommon occurrence, the direct transfer of information from RNA to DNA is thought to be limited to special circumstances. Studies have shown that RNA molecules reverse transcribed into cDNA can be incorporated into DNA at DSB sites via a non-templated mechanism by NHEJ or a templated mechanism by HR. In addition, synthetic RNA molecules can directly template the repair of DSBs in yeast and human cells via an HR mechanism. New work suggests that even endogenous transcript RNA can serve as a homologous template to repair a DSB in chromosomal DNA. In this perspective, we will review and discuss the recent advancements in DSB repair by RNA via non-templated and templated mechanisms. We will provide current findings, models and future challenges investigating RNA and its role in DSB repair.

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“…The galactose inducible promoter of the GAL1 gene (pGal1) can be finely regulated by the amount of inducting agent in the growth medium and by varying the induction time [25,26]. Since VP2 was shown to be non essential for infectivity of the AAV virions [27,28], simultaneous expression of VP1 and VP3 in yeast cells was the principal condition for developing yeast-cell based system for production of the wt like AAV-capsids.…”

Section: Resultsmentioning

confidence: 99%

Capsid protein expression and adeno-associated virus like particles assembly in Saccharomyces cerevisiae

et al. 2012

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BackgroundThe budding yeast Saccharomyces cerevisiae supports replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses) and has provided means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus). We have recently demonstrated that S. cerevisiae can form single stranded DNA AAV2 genomes starting from a circular plasmid. In this work, we have investigated the possibility to assemble AAV capsids in yeast.ResultsTo do this, at least two out of three AAV structural proteins, VP1 and VP3, have to be simultaneously expressed in yeast cells and their intracellular stoichiometry has to resemble the one found in the particles derived from mammalian or insect cells. This was achieved by stable co-transformation of yeast cells with two plasmids, one expressing VP3 from its natural p40 promoter and the other one primarily expressing VP1 from a modified AAV2 Cap gene under the control of the inducible yeast promoter Gal1. Among various induction strategies we tested, the best one to yield the appropriate VP1:VP3 ratio was 4.5 hour induction in the medium containing 0.5% glucose and 5% galactose. Following such induction, AAV virus like particles (VLPs) were isolated from yeast by two step ultracentrifugation procedure. The transmission electron microscopy analysis revealed that their morphology is similar to the empty capsids produced in human cells.ConclusionsTaken together, the results show for the first time that yeast can be used to assemble AAV capsid and, therefore, as a genetic system to identify novel cellular factors involved in AAV biology.

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Ty1 integrase overexpression leads to integration of non-Ty1 DNA fragments into the genome of Saccharomyces cerevisiae

Cited by 10 publications

References 48 publications

The Ty1 LTR-Retrotransposon of Budding Yeast,Saccharomyces cerevisiae

The Ty1 LTR-Retrotransposon of Budding Yeast,Saccharomyces cerevisiae

DNA repair by RNA: Templated, or not templated, that is the question

Capsid protein expression and adeno-associated virus like particles assembly in Saccharomyces cerevisiae

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