Limits...
Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM.

Finney-Manchester SP, Maheshri N - Nucleic Acids Res. (2013)

Bottom Line: By fusing the yeast 3-methyladenine DNA glycosylase MAG1 to a tetR DNA-binding domain, we are able to elevate mutation rates >800 fold in a specific ∼20-kb region of the genome or on a plasmid that contains an array of tetO sites.A wide spectrum of transitions, transversions and single base deletions are observed.We provide evidence that TaGTEAM generated point mutations occur through error-prone homologous recombination (HR) and depend on resectioning and the error-prone polymerase Pol ζ.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

ABSTRACT
A major hurdle to evolutionary engineering approaches for multigenic phenotypes is the ability to simultaneously modify multiple genes rapidly and selectively. Here, we describe a method for in vivo-targeted mutagenesis in yeast, targeting glycosylases to embedded arrays for mutagenesis (TaGTEAM). By fusing the yeast 3-methyladenine DNA glycosylase MAG1 to a tetR DNA-binding domain, we are able to elevate mutation rates >800 fold in a specific ∼20-kb region of the genome or on a plasmid that contains an array of tetO sites. A wide spectrum of transitions, transversions and single base deletions are observed. We provide evidence that TaGTEAM generated point mutations occur through error-prone homologous recombination (HR) and depend on resectioning and the error-prone polymerase Pol ζ. We show that HR is error-prone in this context because of DNA damage checkpoint activation and base pair lesions and use this knowledge to shift the primary mutagenic outcome of targeted endonuclease breaks from HR-independent rearrangements to HR-dependent point mutations. The ability to switch repair in this way opens up the possibility of using targeted endonucleases in diverse organisms for in vivo-targeted mutagenesis.

Show MeSH

Related in: MedlinePlus

Targeted DSBs generated by FokI lead to HR-independent rearrangements and not point mutations. (A) Expression of sctetR-FokI in the same strain background as Mag1-sctetR (B) leads to a similar (620-fold) increase in targeted mutation rates without any increase in background mutation rates. (C) The sctetR-FokI distance dependence is asymmetric, selection for HIS3 leads to a more severe drop in mutation rate, and few PCR+ mutants are generated as compared with Mag1-sctetR. (D) Rad52-CFP repair foci show that sctetR-FokI damage is repaired by HR in roughly the same fraction of cells as Mag1-sctetR, but (E) targeted mutagenesis is not RAD52-dependent, and even under selection for HIS3, there is no REV3-dependence on. Error bars as in Figure 2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3643572&req=5

gkt150-F3: Targeted DSBs generated by FokI lead to HR-independent rearrangements and not point mutations. (A) Expression of sctetR-FokI in the same strain background as Mag1-sctetR (B) leads to a similar (620-fold) increase in targeted mutation rates without any increase in background mutation rates. (C) The sctetR-FokI distance dependence is asymmetric, selection for HIS3 leads to a more severe drop in mutation rate, and few PCR+ mutants are generated as compared with Mag1-sctetR. (D) Rad52-CFP repair foci show that sctetR-FokI damage is repaired by HR in roughly the same fraction of cells as Mag1-sctetR, but (E) targeted mutagenesis is not RAD52-dependent, and even under selection for HIS3, there is no REV3-dependence on. Error bars as in Figure 2.

Mentions: If the sole role of Mag1-sctetR were to create substrates with DNA ends to be repaired by HR, then creating DSBs in the array using an endonuclease might be sufficient for targeted mutagenesis. Although site-specific endonucleases have been associated with neighboring damage (20), such enzymes repeatedly cleave the DNA until mutagenic repair of the recognition site prevents further cleavage. Mag1-sctetR generates significantly fewer Rad52-CFP foci-containing cells than the site-specific HO endonuclease (Figure 2A). To better mimic this infrequent damage at the array, we created a C-terminal fusion of the nuclease domain of FokI to sctetR and expressed it in a strain containing the 240× array and KlURA3 marker at various positions (Figure 3A). We expected lower efficiency cleavage because the monomeric sctetR-FokI must dimerize to be active, and potential partners bound to the array may not be optimally spaced. Similar to Mag1-sctetR, sctetR-FokI elevated the mutation rate at the target 620-fold (Figure 3B). However, sctetR-FokI had no effect on the background mutation rate, either at CAN1 or at KlURA3 in the absence of the array. In addition, sctetR-FokI exhibited an asymmetric distance dependence profile, and few mutants (2/48) were PCR+ (Figure 3C). Although the fraction of cells with Rad52-CFP foci in cells experiencing sctetR-FokI damage at the array was similar to Mag1-sctetR (Figure 3D), RAD52 deletion did not completely eliminate targeted mutagenesis in the absence of HIS3 selection (Figure 3E). Therefore, a large fraction of mutations created by sctetR-FokI are RAD52-independent rearrangements. As expected, this rearrangement did not require the short repetitive sequences present near the array because their elimination using the aforementioned 85× array construct did not decrease the mutation rate (2.7 × 10−5 cell−1 gen−1) and most mutants remained rearrangements (2/12 were PCR+). Under HIS3 selection, mutation rates throughout the target region were further decreased as compared with Mag1-sctetR (Figure 3C). The remaining mutagenesis was independent of Pol ζ (Figure 3E) and still predominantly rearrangements (8/32 were PCR+). Therefore, processing of FokI-generated damage—presumably DSBs—results in loss of KlURA3 function largely through RAD52-independent rearrangements rather than the LHM process in Figure 2B.Figure 3.


Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM.

Finney-Manchester SP, Maheshri N - Nucleic Acids Res. (2013)

Targeted DSBs generated by FokI lead to HR-independent rearrangements and not point mutations. (A) Expression of sctetR-FokI in the same strain background as Mag1-sctetR (B) leads to a similar (620-fold) increase in targeted mutation rates without any increase in background mutation rates. (C) The sctetR-FokI distance dependence is asymmetric, selection for HIS3 leads to a more severe drop in mutation rate, and few PCR+ mutants are generated as compared with Mag1-sctetR. (D) Rad52-CFP repair foci show that sctetR-FokI damage is repaired by HR in roughly the same fraction of cells as Mag1-sctetR, but (E) targeted mutagenesis is not RAD52-dependent, and even under selection for HIS3, there is no REV3-dependence on. Error bars as in Figure 2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643572&req=5

gkt150-F3: Targeted DSBs generated by FokI lead to HR-independent rearrangements and not point mutations. (A) Expression of sctetR-FokI in the same strain background as Mag1-sctetR (B) leads to a similar (620-fold) increase in targeted mutation rates without any increase in background mutation rates. (C) The sctetR-FokI distance dependence is asymmetric, selection for HIS3 leads to a more severe drop in mutation rate, and few PCR+ mutants are generated as compared with Mag1-sctetR. (D) Rad52-CFP repair foci show that sctetR-FokI damage is repaired by HR in roughly the same fraction of cells as Mag1-sctetR, but (E) targeted mutagenesis is not RAD52-dependent, and even under selection for HIS3, there is no REV3-dependence on. Error bars as in Figure 2.
Mentions: If the sole role of Mag1-sctetR were to create substrates with DNA ends to be repaired by HR, then creating DSBs in the array using an endonuclease might be sufficient for targeted mutagenesis. Although site-specific endonucleases have been associated with neighboring damage (20), such enzymes repeatedly cleave the DNA until mutagenic repair of the recognition site prevents further cleavage. Mag1-sctetR generates significantly fewer Rad52-CFP foci-containing cells than the site-specific HO endonuclease (Figure 2A). To better mimic this infrequent damage at the array, we created a C-terminal fusion of the nuclease domain of FokI to sctetR and expressed it in a strain containing the 240× array and KlURA3 marker at various positions (Figure 3A). We expected lower efficiency cleavage because the monomeric sctetR-FokI must dimerize to be active, and potential partners bound to the array may not be optimally spaced. Similar to Mag1-sctetR, sctetR-FokI elevated the mutation rate at the target 620-fold (Figure 3B). However, sctetR-FokI had no effect on the background mutation rate, either at CAN1 or at KlURA3 in the absence of the array. In addition, sctetR-FokI exhibited an asymmetric distance dependence profile, and few mutants (2/48) were PCR+ (Figure 3C). Although the fraction of cells with Rad52-CFP foci in cells experiencing sctetR-FokI damage at the array was similar to Mag1-sctetR (Figure 3D), RAD52 deletion did not completely eliminate targeted mutagenesis in the absence of HIS3 selection (Figure 3E). Therefore, a large fraction of mutations created by sctetR-FokI are RAD52-independent rearrangements. As expected, this rearrangement did not require the short repetitive sequences present near the array because their elimination using the aforementioned 85× array construct did not decrease the mutation rate (2.7 × 10−5 cell−1 gen−1) and most mutants remained rearrangements (2/12 were PCR+). Under HIS3 selection, mutation rates throughout the target region were further decreased as compared with Mag1-sctetR (Figure 3C). The remaining mutagenesis was independent of Pol ζ (Figure 3E) and still predominantly rearrangements (8/32 were PCR+). Therefore, processing of FokI-generated damage—presumably DSBs—results in loss of KlURA3 function largely through RAD52-independent rearrangements rather than the LHM process in Figure 2B.Figure 3.

Bottom Line: By fusing the yeast 3-methyladenine DNA glycosylase MAG1 to a tetR DNA-binding domain, we are able to elevate mutation rates >800 fold in a specific ∼20-kb region of the genome or on a plasmid that contains an array of tetO sites.A wide spectrum of transitions, transversions and single base deletions are observed.We provide evidence that TaGTEAM generated point mutations occur through error-prone homologous recombination (HR) and depend on resectioning and the error-prone polymerase Pol ζ.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

ABSTRACT
A major hurdle to evolutionary engineering approaches for multigenic phenotypes is the ability to simultaneously modify multiple genes rapidly and selectively. Here, we describe a method for in vivo-targeted mutagenesis in yeast, targeting glycosylases to embedded arrays for mutagenesis (TaGTEAM). By fusing the yeast 3-methyladenine DNA glycosylase MAG1 to a tetR DNA-binding domain, we are able to elevate mutation rates >800 fold in a specific ∼20-kb region of the genome or on a plasmid that contains an array of tetO sites. A wide spectrum of transitions, transversions and single base deletions are observed. We provide evidence that TaGTEAM generated point mutations occur through error-prone homologous recombination (HR) and depend on resectioning and the error-prone polymerase Pol ζ. We show that HR is error-prone in this context because of DNA damage checkpoint activation and base pair lesions and use this knowledge to shift the primary mutagenic outcome of targeted endonuclease breaks from HR-independent rearrangements to HR-dependent point mutations. The ability to switch repair in this way opens up the possibility of using targeted endonucleases in diverse organisms for in vivo-targeted mutagenesis.

Show MeSH
Related in: MedlinePlus