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The fate of linear DNA in Saccharomyces cerevisiae and Candida glabrata: the role of homologous and non-homologous end joining.

Corrigan MW, Kerwin-Iosue CL, Kuczmarski AS, Amin KB, Wykoff DD - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Villanova University, Villanova, Pennsylvania, United States.

ABSTRACT
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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Model of the fate of linear DNA in S. cerevisiae and C. glabrata.In S. cerevisiae, we were unable to observe re-circularization via NHEJ; however, it is likely that there is still a low level of NHEJ activity. Consequently, there is little change in the frequency of HR in both S. cerevisiae wild-type and dnl4Δ. In contrast, in C. glabrata, we observe insertion and vector ligation via NHEJ. Deletion of DNL4 results in an increase in HR, indicating that NHEJ is more prevalent in C. glabrata.
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pone-0069628-g007: Model of the fate of linear DNA in S. cerevisiae and C. glabrata.In S. cerevisiae, we were unable to observe re-circularization via NHEJ; however, it is likely that there is still a low level of NHEJ activity. Consequently, there is little change in the frequency of HR in both S. cerevisiae wild-type and dnl4Δ. In contrast, in C. glabrata, we observe insertion and vector ligation via NHEJ. Deletion of DNL4 results in an increase in HR, indicating that NHEJ is more prevalent in C. glabrata.

Mentions: Based on results from all of the experiments that were performed, we generated a model for the fate of linear DNA in S. cerevisiae and C. glabrata (Figure 7). In S. cerevisiae, it appears that the linear DNA (both pRS313 and PCR product) takes two out of three avenues in the majority of instances: HR or vector degradation (the eventual fate of transformed linear DNA in cells if vector is not re-circularized). Removing NHEJ activity in S. cerevisiae, through deletion of DNL4, has little effect on GRC, because there is little NHEJ to begin with. On the other hand, the fate of linear DNA in C. glabrata is influenced by HR, NHEJ, or degradation. When NHEJ activity is deleted in C. glabrata, there is an increase in HR. Although both species are capable of performing GRC at a high level, this model suggests that S. cerevisiae is the preferred species in which to perform this technique. Our data indicate that NHEJ is more prevalent in C. glabrata; however we cannot fully determine whether this is because NHEJ is inherently more active in this species, or whether linear DNA is degraded more slowly in C. glabrata and the NHEJ machinery is able to work for a longer period.


The fate of linear DNA in Saccharomyces cerevisiae and Candida glabrata: the role of homologous and non-homologous end joining.

Corrigan MW, Kerwin-Iosue CL, Kuczmarski AS, Amin KB, Wykoff DD - PLoS ONE (2013)

Model of the fate of linear DNA in S. cerevisiae and C. glabrata.In S. cerevisiae, we were unable to observe re-circularization via NHEJ; however, it is likely that there is still a low level of NHEJ activity. Consequently, there is little change in the frequency of HR in both S. cerevisiae wild-type and dnl4Δ. In contrast, in C. glabrata, we observe insertion and vector ligation via NHEJ. Deletion of DNL4 results in an increase in HR, indicating that NHEJ is more prevalent in C. glabrata.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3722132&req=5

pone-0069628-g007: Model of the fate of linear DNA in S. cerevisiae and C. glabrata.In S. cerevisiae, we were unable to observe re-circularization via NHEJ; however, it is likely that there is still a low level of NHEJ activity. Consequently, there is little change in the frequency of HR in both S. cerevisiae wild-type and dnl4Δ. In contrast, in C. glabrata, we observe insertion and vector ligation via NHEJ. Deletion of DNL4 results in an increase in HR, indicating that NHEJ is more prevalent in C. glabrata.
Mentions: Based on results from all of the experiments that were performed, we generated a model for the fate of linear DNA in S. cerevisiae and C. glabrata (Figure 7). In S. cerevisiae, it appears that the linear DNA (both pRS313 and PCR product) takes two out of three avenues in the majority of instances: HR or vector degradation (the eventual fate of transformed linear DNA in cells if vector is not re-circularized). Removing NHEJ activity in S. cerevisiae, through deletion of DNL4, has little effect on GRC, because there is little NHEJ to begin with. On the other hand, the fate of linear DNA in C. glabrata is influenced by HR, NHEJ, or degradation. When NHEJ activity is deleted in C. glabrata, there is an increase in HR. Although both species are capable of performing GRC at a high level, this model suggests that S. cerevisiae is the preferred species in which to perform this technique. Our data indicate that NHEJ is more prevalent in C. glabrata; however we cannot fully determine whether this is because NHEJ is inherently more active in this species, or whether linear DNA is degraded more slowly in C. glabrata and the NHEJ machinery is able to work for a longer period.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Villanova University, Villanova, Pennsylvania, United States.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus