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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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A model for the mutagenic outcome at KlURA3 because of targeted damage generated by Mag1-sctetR or sctetR-FokI. Mag1-sctetR and sctetR-FokI both generate lesions that lead to DSBs, but the mutagenic repair outcome at KlURA3 depends on two conditions: (i) checkpoint activation and (ii) base pair damage. Repair that is error-free at KlURA3 and cell death are other possible outcomes of targeted damage. (A) In cells expressing Mag1-sctetR, both conditions are present, leading to high rate point mutagenesis and minimal HR-independent rearrangement. (B) sctetR-FokI expressing cells do not activate the DNA damage checkpoint to the same extent or experience base pair damage and the primary mutagenic event is HR-independent rearrangement. Co-expression of untargeted Mag1p with sctetR-FokI or addition of MMS (C) mimics Mag1-sctetR mutagenesis. Addition of 4.5 mg/ml HU (D) demonstrates that the transition in primary mutagenic outcome from HR-independent rearrangements to HR- and REV3-dependent point mutations occurs only when both conditions are met.
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gkt150-F5: A model for the mutagenic outcome at KlURA3 because of targeted damage generated by Mag1-sctetR or sctetR-FokI. Mag1-sctetR and sctetR-FokI both generate lesions that lead to DSBs, but the mutagenic repair outcome at KlURA3 depends on two conditions: (i) checkpoint activation and (ii) base pair damage. Repair that is error-free at KlURA3 and cell death are other possible outcomes of targeted damage. (A) In cells expressing Mag1-sctetR, both conditions are present, leading to high rate point mutagenesis and minimal HR-independent rearrangement. (B) sctetR-FokI expressing cells do not activate the DNA damage checkpoint to the same extent or experience base pair damage and the primary mutagenic event is HR-independent rearrangement. Co-expression of untargeted Mag1p with sctetR-FokI or addition of MMS (C) mimics Mag1-sctetR mutagenesis. Addition of 4.5 mg/ml HU (D) demonstrates that the transition in primary mutagenic outcome from HR-independent rearrangements to HR- and REV3-dependent point mutations occurs only when both conditions are met.

Mentions: Combining our analysis of Mag1-sctetR– and sctetR-FokI–generated damage suggests that genome-wide DNA damage and DNA damage checkpoint activation serve as two distinct control points that switch the primary mutagenic outcome resulting from clustered DNA damage (Figure 5). When applied to endonuclease-derived breaks, these control points allow for the downregulation of HR-independent rearrangements (checkpoint activation with HU) and the upregulation of point mutagenesis (genome-wide DNA damage with MMS). It remains unclear which control point is rate limiting in generating point mutations. Even though the targeted mutation rate plateaus with increasing levels of MMS (Figure 4D), this may be due to growth defects rather than saturating ssDNA lesions. Moreover, the use of HU leads to activation of an intra-S checkpoint rather than a G2/M checkpoint. Because both these checkpoints are Mec1p-dependent and require DDC2 (43), we have shown that lack of checkpoint activation decreases HR-dependent point mutations. Whether greater checkpoint activation or arrest in S versus G2/M affects the number of breaks undergo extensive resectioning, or if ssDNA-specific damaging agents can lead to multiple point mutations per lesion event will be the subject of future work.Figure 5.


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

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

A model for the mutagenic outcome at KlURA3 because of targeted damage generated by Mag1-sctetR or sctetR-FokI. Mag1-sctetR and sctetR-FokI both generate lesions that lead to DSBs, but the mutagenic repair outcome at KlURA3 depends on two conditions: (i) checkpoint activation and (ii) base pair damage. Repair that is error-free at KlURA3 and cell death are other possible outcomes of targeted damage. (A) In cells expressing Mag1-sctetR, both conditions are present, leading to high rate point mutagenesis and minimal HR-independent rearrangement. (B) sctetR-FokI expressing cells do not activate the DNA damage checkpoint to the same extent or experience base pair damage and the primary mutagenic event is HR-independent rearrangement. Co-expression of untargeted Mag1p with sctetR-FokI or addition of MMS (C) mimics Mag1-sctetR mutagenesis. Addition of 4.5 mg/ml HU (D) demonstrates that the transition in primary mutagenic outcome from HR-independent rearrangements to HR- and REV3-dependent point mutations occurs only when both conditions are met.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt150-F5: A model for the mutagenic outcome at KlURA3 because of targeted damage generated by Mag1-sctetR or sctetR-FokI. Mag1-sctetR and sctetR-FokI both generate lesions that lead to DSBs, but the mutagenic repair outcome at KlURA3 depends on two conditions: (i) checkpoint activation and (ii) base pair damage. Repair that is error-free at KlURA3 and cell death are other possible outcomes of targeted damage. (A) In cells expressing Mag1-sctetR, both conditions are present, leading to high rate point mutagenesis and minimal HR-independent rearrangement. (B) sctetR-FokI expressing cells do not activate the DNA damage checkpoint to the same extent or experience base pair damage and the primary mutagenic event is HR-independent rearrangement. Co-expression of untargeted Mag1p with sctetR-FokI or addition of MMS (C) mimics Mag1-sctetR mutagenesis. Addition of 4.5 mg/ml HU (D) demonstrates that the transition in primary mutagenic outcome from HR-independent rearrangements to HR- and REV3-dependent point mutations occurs only when both conditions are met.
Mentions: Combining our analysis of Mag1-sctetR– and sctetR-FokI–generated damage suggests that genome-wide DNA damage and DNA damage checkpoint activation serve as two distinct control points that switch the primary mutagenic outcome resulting from clustered DNA damage (Figure 5). When applied to endonuclease-derived breaks, these control points allow for the downregulation of HR-independent rearrangements (checkpoint activation with HU) and the upregulation of point mutagenesis (genome-wide DNA damage with MMS). It remains unclear which control point is rate limiting in generating point mutations. Even though the targeted mutation rate plateaus with increasing levels of MMS (Figure 4D), this may be due to growth defects rather than saturating ssDNA lesions. Moreover, the use of HU leads to activation of an intra-S checkpoint rather than a G2/M checkpoint. Because both these checkpoints are Mec1p-dependent and require DDC2 (43), we have shown that lack of checkpoint activation decreases HR-dependent point mutations. Whether greater checkpoint activation or arrest in S versus G2/M affects the number of breaks undergo extensive resectioning, or if ssDNA-specific damaging agents can lead to multiple point mutations per lesion event will be the subject of future work.Figure 5.

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