Influence of homology size and polymorphism on plasmid integration in the yeast CYC1 DNA region

Koren, Predrag; Svetec, Ivan‐Krešimir; Mitrikeski, Petar Tomev; Zgaga, Zoran

doi:10.1007/s002940050530

Cited by 10 publications

(11 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Therefore, efficient single stranded DNA assimilation in the recombination intermediate, followed by a strong antirecombinogenic effect of 4 bp heterology could result in the frequent displacement of the assimilated single stranded DNA and an appearance of a single stranded transforming DNA fragment. This is in agreement with the previous reports showing that single stranded DNA is highly recombinogenic, being less affected by antirecombinogenic effect of point mutations [77], prone to illegitimate recombination and able to induce genome rearrangement [53]. …”

Section: Discussionsupporting

confidence: 93%

Sgs1 and Exo1 suppress targeted chromosome duplication during ends-in and ends-out gene targeting

Štafa¹,

Miklenić²,

Žunar³

et al. 2014

DNA Repair

View full text Add to dashboard Cite

Gene targeting is extremely efficient in the yeast Saccharomyces cerevisiae. It is performed by transformation with a linear, non-replicative DNA fragment carrying a selectable marker and containing ends homologous to the particular locus in a genome. However, even in S. cerevisiae, transformation can result in unwanted (aberrant) integration events, the frequency and spectra of which are quite different for ends-out and ends-in transformation assays. It has been observed that gene replacement (ends-out gene targeting) can result in illegitimate integration, integration of the transforming DNA fragment next to the target sequence and duplication of a targeted chromosome. By contrast, plasmid integration (ends-in gene targeting) is often associated with multiple targeted integration events but illegitimate integration is extremely rare and a targeted chromosome duplication has not been reported. Here we systematically investigated the influence of design of the ends-out assay on the success of targeted genetic modification. We have determined transformation efficiency, fidelity of gene targeting and spectra of all aberrant events in several ends-out gene targeting assays designed to insert, delete or replace a particular sequence in the targeted region of the yeast genome. Furthermore, we have demonstrated for the first time that targeted chromosome duplications occur even during ends-in gene targeting. Most importantly, the whole chromosome duplication is POL32 dependent pointing to break-induced replication (BIR) as the underlying mechanism. Moreover, the occurrence of duplication of the targeted chromosome was strikingly increased in the exo1Δ sgs1Δ double mutant but not in the respective single mutants demonstrating that the Exo1 and Sgs1 proteins independently suppress whole chromosome duplication during gene targeting.

show abstract

Section: Discussionsupporting

confidence: 93%

Sgs1 and Exo1 suppress targeted chromosome duplication during ends-in and ends-out gene targeting

Štafa¹,

Miklenić²,

Žunar³

et al. 2014

DNA Repair

View full text Add to dashboard Cite

show abstract

“…Another fact supports the hypothesis that plasmids hit their homologous target region randomly: It is the correlation between the homologous integration efficiency and the length of the p:target region, that was observed by several authors [147,150,151]. The larger the p:target region the higher the probability that the vector hits the homologous c:target region.…”

Section: The Pathway Of the Vector Through The Cellsupporting

confidence: 74%

“…The Rad51 strand transfer protein behaves in a very stringent way and tolerates only heterologies that do not exceed 9 bp [160]. Integration frequency of circular plasmids is lowered by sequence mismatches between the p:target and the c:target [151]. Disruption of the mismatch repair gene MSH2 enhances recombination between mismatched DNA targets [158].…”

Section: Possible Rate‐limiting Stepsmentioning

confidence: 99%

Genetic aspects of targeted insertion mutagenesis in yeasts

Klinner

Schäfer

2004

FEMS Microbiol Rev

View full text Add to dashboard Cite

Targeted insertion mutagenesis is a main molecular tool of yeast science initially applied in Saccharomyces cerevisiae. The method was extended to fission yeast Schizosaccharomyces pombe and to "non-conventional" yeast species, which show specific properties of special interest to both basic and applied research. Consequently, the behaviour of such non-Saccharomyces yeasts is reviewed against the background of the knowledge of targeted insertion mutagenesis in S. cerevisiae. Data of homologous integration efficiencies obtained with circular, ends-in or ends-out vectors in several yeasts are compared. We follow details of targeted insertion mutagenesis in order to recognize possible rate-limiting steps. The route of the vector to the target and possible mechanisms of its integration into chromosomal genes are considered. Specific features of some yeast species are discussed. In addition, similar approaches based on homologous recombination that have been established for the mitochondrial genome of S. cerevisiae are described.

show abstract

“…This interpretation is supported by other findings that large heterologies are efficiently introduced into heteroduplex DNA, both in bacteria and yeast (Adams and West 1996;Clikeman et al 2001). Their conversion during plasmid integration in the yeast genome is rare, occurring in only $1% of repair events (Koren et al 2000). Therefore, by combining the results of the replica-plating and the corresponding colony PCR, we consider that the fraction of transformants showing unexpected phenotypes indeed reflects the incidence of integration events into DNA regions distant from the site of the induced DSB.…”

Section: Resultsmentioning

confidence: 99%

“…Using PCR disruption cassettes for systematic disruption of yeast ORFs, many loci have been found to be very difficult to disrupt due to the low efficiency of integration of the disruption cassette, forcing the development of new knockout strategies (Puig et al 1998;Nikawa and Kawabata 1998;Lucau-Danila et al 2000). Similarly, several studies of the mechanism of homologous recombination, using integrative plasmids bearing two yeast DNA fragments of similar size, have demonstrated a preference for one or other of the homologous genomic loci (Orr-Weaver et al 1981;Ninkovic et al 1994;Koren et al 2000). Despite these intriguing observations, no in-depth study has yet been performed to discover the reasons for differences in the frequency of integrative transformation, often referred as locusdependent recombination.…”

Section: Introductionmentioning

confidence: 98%

Targeted DNA integration within different functional gene domains in yeast reveals ORF sequences as recombinational cold-spots

2004

View full text Add to dashboard Cite

The efficiency of gene targeting within different segments of genes in yeast was estimated by transforming yeast cells with double-stranded integrative plasmids, bearing functional gene domains [promoter (P), ORF (O) and terminator (T)] derived from the common genetic markers HIS3, LEU2, TRP1 and URA3. Transformation experiments with circular plasmids carrying a single gene domain demonstrated that the 5' and 3' flanking DNA regions (P and T) of the HIS3 and URA3 genes are preferred as sites for plasmid integration by several fold over the corresponding ORFs. Moreover, when plasmids bearing combinations of two or three regions were linearized to target them to a specific site of integration, three of the ORFs were found to be less preferred as sites for plasmid integration than their corresponding flanking regions. Surprisingly, in up to 50% of the transformants obtained with plasmids that had been linearized within coding sequences, the DNA actually integrated into neighbouring regions. Almost the same frequencies of ORF mis-targeting were obtained with plasmid vectors containing only two functional domains ("PO" or "OT") of the gene URA3, demonstrating that this event is not the consequence of competition between homologous DNA regions distal to the ORF. Therefore, we suggest that coding sequences could be considered to be "cold spots" for plasmid integration in yeast.

show abstract

Influence of homology size and polymorphism on plasmid integration in the yeast CYC1 DNA region

Cited by 10 publications

References 44 publications

Sgs1 and Exo1 suppress targeted chromosome duplication during ends-in and ends-out gene targeting

Sgs1 and Exo1 suppress targeted chromosome duplication during ends-in and ends-out gene targeting

Genetic aspects of targeted insertion mutagenesis in yeasts

Targeted DNA integration within different functional gene domains in yeast reveals ORF sequences as recombinational cold-spots

Contact Info

Product

Resources

About