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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.

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Global DNA damage redirects mutagenic repair of sctetR-FokI–induced breaks towards HR-dependent point mutations via checkpoint activation and DNA lesions. Mutation rates generated by sctetR-FokI expression in WT, Pol ζ-deficient (rev3) and checkpoint-deficient (sml1 ddc2) strains were measured and compared with those in the presence of co-expressed Mag1p, MMS or HU. (A) In the absence of selection for HIS3, co-expression of Mag1p with sctetR-FokI makes checkpoint- and Pol ζ-dependent point mutagenesis the dominant mutagenic outcome, as indicated by scoring of mutants for a His+ and/or PCR+ phenotype (listed above bars). HU, on the other hand, decreases HR-independent rearrangements without creating point mutations. In the absence of Mag1 activity, loss of checkpoint activation leads to very high (>10−4 cell−1 gen−1) mutation rates that correspond to rearrangements. (B) HIS3 selection reveals Pol ζ-dependent point mutations generated by the addition of MMS. (C) Overnight growth of cells in various levels of MMS compared with growth without MMS. (D) Mutation rates in cells expressing sctetR-FokI reach a maximum at 0.003% MMS. Selection for HIS3 reveals that the majority of mutations at this level of MMS are point mutations. In every case observed, His− mutants were never PCR+. Addition of HU to sml1 ddc2 strains eliminates growth, preventing measurement of the mutation rate. Error bars are 95% CI.
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gkt150-F4: Global DNA damage redirects mutagenic repair of sctetR-FokI–induced breaks towards HR-dependent point mutations via checkpoint activation and DNA lesions. Mutation rates generated by sctetR-FokI expression in WT, Pol ζ-deficient (rev3) and checkpoint-deficient (sml1 ddc2) strains were measured and compared with those in the presence of co-expressed Mag1p, MMS or HU. (A) In the absence of selection for HIS3, co-expression of Mag1p with sctetR-FokI makes checkpoint- and Pol ζ-dependent point mutagenesis the dominant mutagenic outcome, as indicated by scoring of mutants for a His+ and/or PCR+ phenotype (listed above bars). HU, on the other hand, decreases HR-independent rearrangements without creating point mutations. In the absence of Mag1 activity, loss of checkpoint activation leads to very high (>10−4 cell−1 gen−1) mutation rates that correspond to rearrangements. (B) HIS3 selection reveals Pol ζ-dependent point mutations generated by the addition of MMS. (C) Overnight growth of cells in various levels of MMS compared with growth without MMS. (D) Mutation rates in cells expressing sctetR-FokI reach a maximum at 0.003% MMS. Selection for HIS3 reveals that the majority of mutations at this level of MMS are point mutations. In every case observed, His− mutants were never PCR+. Addition of HU to sml1 ddc2 strains eliminates growth, preventing measurement of the mutation rate. Error bars are 95% CI.

Mentions: As sctetR-FokI damage increases Rad52 foci (Figure 3D), much of it must be repaired via HR without mutating KlURA3. Understanding why these HR repair events do not lead to point mutations and why the dominant mutagenic event is RAD52-independent rearrangements could allow us to increase point mutations and potentially use any DSB to generate them. We hypothesized that differences between sctetR-FokI and Mag1-sctetR were either because of the nature of the break intermediate or the cellular context in which the break was repaired. In support of the second hypothesis, Mag1-sctetR, but not sctetR-FokI, has a non-specific DNA damaging activity that increases background mutation rates (Figure 1B and C) and increases the fraction of cells with Rad52-CFP foci in the absence of the array (Figures 2B and 3D). To test whether the non-specific DNA damage activity of Mag1-sctetR explains the difference in types of mutations generated by each mutator, we co-expressed untargeted Mag1p with sctetR-FokI (Figure 4). Mag1p co-expression was sufficient to switch mutations generated by sctetR-FokI to predominantly point mutations (11/12 were PCR+). HIS3 selection caused no drop in the observed mutation rate, and like Mag1-sctetR, targeted mutagenesis was REV3-dependent. The mutation spectrum was also similar to Mag1-sctetR (Table 1), consistent with mutations occurring at bases damaged by Mag1.Figure 4.


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

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

Global DNA damage redirects mutagenic repair of sctetR-FokI–induced breaks towards HR-dependent point mutations via checkpoint activation and DNA lesions. Mutation rates generated by sctetR-FokI expression in WT, Pol ζ-deficient (rev3) and checkpoint-deficient (sml1 ddc2) strains were measured and compared with those in the presence of co-expressed Mag1p, MMS or HU. (A) In the absence of selection for HIS3, co-expression of Mag1p with sctetR-FokI makes checkpoint- and Pol ζ-dependent point mutagenesis the dominant mutagenic outcome, as indicated by scoring of mutants for a His+ and/or PCR+ phenotype (listed above bars). HU, on the other hand, decreases HR-independent rearrangements without creating point mutations. In the absence of Mag1 activity, loss of checkpoint activation leads to very high (>10−4 cell−1 gen−1) mutation rates that correspond to rearrangements. (B) HIS3 selection reveals Pol ζ-dependent point mutations generated by the addition of MMS. (C) Overnight growth of cells in various levels of MMS compared with growth without MMS. (D) Mutation rates in cells expressing sctetR-FokI reach a maximum at 0.003% MMS. Selection for HIS3 reveals that the majority of mutations at this level of MMS are point mutations. In every case observed, His− mutants were never PCR+. Addition of HU to sml1 ddc2 strains eliminates growth, preventing measurement of the mutation rate. Error bars are 95% CI.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt150-F4: Global DNA damage redirects mutagenic repair of sctetR-FokI–induced breaks towards HR-dependent point mutations via checkpoint activation and DNA lesions. Mutation rates generated by sctetR-FokI expression in WT, Pol ζ-deficient (rev3) and checkpoint-deficient (sml1 ddc2) strains were measured and compared with those in the presence of co-expressed Mag1p, MMS or HU. (A) In the absence of selection for HIS3, co-expression of Mag1p with sctetR-FokI makes checkpoint- and Pol ζ-dependent point mutagenesis the dominant mutagenic outcome, as indicated by scoring of mutants for a His+ and/or PCR+ phenotype (listed above bars). HU, on the other hand, decreases HR-independent rearrangements without creating point mutations. In the absence of Mag1 activity, loss of checkpoint activation leads to very high (>10−4 cell−1 gen−1) mutation rates that correspond to rearrangements. (B) HIS3 selection reveals Pol ζ-dependent point mutations generated by the addition of MMS. (C) Overnight growth of cells in various levels of MMS compared with growth without MMS. (D) Mutation rates in cells expressing sctetR-FokI reach a maximum at 0.003% MMS. Selection for HIS3 reveals that the majority of mutations at this level of MMS are point mutations. In every case observed, His− mutants were never PCR+. Addition of HU to sml1 ddc2 strains eliminates growth, preventing measurement of the mutation rate. Error bars are 95% CI.
Mentions: As sctetR-FokI damage increases Rad52 foci (Figure 3D), much of it must be repaired via HR without mutating KlURA3. Understanding why these HR repair events do not lead to point mutations and why the dominant mutagenic event is RAD52-independent rearrangements could allow us to increase point mutations and potentially use any DSB to generate them. We hypothesized that differences between sctetR-FokI and Mag1-sctetR were either because of the nature of the break intermediate or the cellular context in which the break was repaired. In support of the second hypothesis, Mag1-sctetR, but not sctetR-FokI, has a non-specific DNA damaging activity that increases background mutation rates (Figure 1B and C) and increases the fraction of cells with Rad52-CFP foci in the absence of the array (Figures 2B and 3D). To test whether the non-specific DNA damage activity of Mag1-sctetR explains the difference in types of mutations generated by each mutator, we co-expressed untargeted Mag1p with sctetR-FokI (Figure 4). Mag1p co-expression was sufficient to switch mutations generated by sctetR-FokI to predominantly point mutations (11/12 were PCR+). HIS3 selection caused no drop in the observed mutation rate, and like Mag1-sctetR, targeted mutagenesis was REV3-dependent. The mutation spectrum was also similar to Mag1-sctetR (Table 1), consistent with mutations occurring at bases damaged by Mag1.Figure 4.

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