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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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TaGTEAM increases the mutation rate in a 20-kb region surrounding the tetO array. (A) The Mag1-sctetR fusion is expressed from a galactose-inducible promoter on a centromeric plasmid present in cells containing a 9-kb 240× tetO array integrated on the right arm of chromosome I. All distances are relative to the nearest edge of the tetO array, and KlURA3 markers indicted by dotted boxes show alternative integration sites used for measuring the distance dependence. (B) With an integrated tetO array and Mag1-sctetR expression, mutation rates at the 0.3 kb target are elevated 800-fold as measured by fluctuation assays, whereas rates at the CAN1 marker on chromosome V do not change. (C) This increase in mutation rate persists for at least 10 kb on either side of the array as measured in strains with KlURA3 integrated the specified distance from the tetO array (one instance per strain). Selection for HIS3 (diamonds) decreases the mutation rate slightly and addition of dox (squares) eliminates targeted mutagenesis completely. (D) TaGTEAM also functions when targeted to a plasmid containing the tetO array. Mutation rates were monitored using the gain of function marker ade2-1, which reverts through base pair substitutions at an internal stop codon. Labels on data points report the ability to PCR KlURA3 from a particular mutant, PCR+(total). Error bars are 95% CI.
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gkt150-F1: TaGTEAM increases the mutation rate in a 20-kb region surrounding the tetO array. (A) The Mag1-sctetR fusion is expressed from a galactose-inducible promoter on a centromeric plasmid present in cells containing a 9-kb 240× tetO array integrated on the right arm of chromosome I. All distances are relative to the nearest edge of the tetO array, and KlURA3 markers indicted by dotted boxes show alternative integration sites used for measuring the distance dependence. (B) With an integrated tetO array and Mag1-sctetR expression, mutation rates at the 0.3 kb target are elevated 800-fold as measured by fluctuation assays, whereas rates at the CAN1 marker on chromosome V do not change. (C) This increase in mutation rate persists for at least 10 kb on either side of the array as measured in strains with KlURA3 integrated the specified distance from the tetO array (one instance per strain). Selection for HIS3 (diamonds) decreases the mutation rate slightly and addition of dox (squares) eliminates targeted mutagenesis completely. (D) TaGTEAM also functions when targeted to a plasmid containing the tetO array. Mutation rates were monitored using the gain of function marker ade2-1, which reverts through base pair substitutions at an internal stop codon. Labels on data points report the ability to PCR KlURA3 from a particular mutant, PCR+(total). Error bars are 95% CI.

Mentions: We chose to use a DNA glycosylase as our mutator enzyme and localize it by fusion with the tet repressor (tetR) that binds the 19-bp tet operator (tetO) sequence. DNA glycosylases normally function as the first step in base excision repair (BER) to remove chemically altered DNA bases. A build-up of unprocessed abasic sites leads to replication fork stalling and recruitment of error-prone trans-lesion polymerases (26). This faulty repair can lead to both point mutations and frameshifts (27). We tested the yeast 3-methyladenine glycosylase Mag1p, which is primarily responsible for excising alkylated bases, but has naturally broad substrate specificity (28) and is thought to excise normal base pairs when overexpressed (29). We fused Mag1p to single-chain (sc) tetR. Normal tetR forms a dimer; therefore, fusion to tetR may result in impaired enzymatic or binding activity because of a loss of conformational freedom caused by having two copies of Mag1p in such close proximity. Fusion of Mag1 to sctetR—a tandem repeat of tetR connected via a peptide linker (30)—results in one copy of Mag1 per tetR dimer and eliminates this problem (see Figure 1A). We then verified the mutator activity of Mag1-sctetR by overexpressing it in an apn1Δ background, as reduced AP (apurinic/apyrimidinic) endonuclease activity elevates mutation rates (29) (Supplementary Figure S1). Finally, we introduced a tetR-repressible promoter driving yeast fluorescent protein (YFP) (31) in cells expressing Mag1-sctetR to confirm it bound tetO sites. When doxycycline (dox) is added, it binds to and reduces the affinity of sctetR for tetO, relieving the repression and increasing YFP expression. Mag1-sctetR repressed YFP expression to nearly the same extent as sctetR (Supplementary Figure S1E). Therefore, Mag1-sctetR has both mutagenic and specific DNA-binding activity. Similar experiments using cytosine DNA glycosylase (CDG), a mutant of human uracil DNA glycosylase specific for excising cytosines (32), did not yield targeted mutagenesis, probably because of the low expression of the CDG–sctetR fusion (Supplementary Figures S1 and S2).Figure 1.


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

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

TaGTEAM increases the mutation rate in a 20-kb region surrounding the tetO array. (A) The Mag1-sctetR fusion is expressed from a galactose-inducible promoter on a centromeric plasmid present in cells containing a 9-kb 240× tetO array integrated on the right arm of chromosome I. All distances are relative to the nearest edge of the tetO array, and KlURA3 markers indicted by dotted boxes show alternative integration sites used for measuring the distance dependence. (B) With an integrated tetO array and Mag1-sctetR expression, mutation rates at the 0.3 kb target are elevated 800-fold as measured by fluctuation assays, whereas rates at the CAN1 marker on chromosome V do not change. (C) This increase in mutation rate persists for at least 10 kb on either side of the array as measured in strains with KlURA3 integrated the specified distance from the tetO array (one instance per strain). Selection for HIS3 (diamonds) decreases the mutation rate slightly and addition of dox (squares) eliminates targeted mutagenesis completely. (D) TaGTEAM also functions when targeted to a plasmid containing the tetO array. Mutation rates were monitored using the gain of function marker ade2-1, which reverts through base pair substitutions at an internal stop codon. Labels on data points report the ability to PCR KlURA3 from a particular mutant, PCR+(total). Error bars are 95% CI.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt150-F1: TaGTEAM increases the mutation rate in a 20-kb region surrounding the tetO array. (A) The Mag1-sctetR fusion is expressed from a galactose-inducible promoter on a centromeric plasmid present in cells containing a 9-kb 240× tetO array integrated on the right arm of chromosome I. All distances are relative to the nearest edge of the tetO array, and KlURA3 markers indicted by dotted boxes show alternative integration sites used for measuring the distance dependence. (B) With an integrated tetO array and Mag1-sctetR expression, mutation rates at the 0.3 kb target are elevated 800-fold as measured by fluctuation assays, whereas rates at the CAN1 marker on chromosome V do not change. (C) This increase in mutation rate persists for at least 10 kb on either side of the array as measured in strains with KlURA3 integrated the specified distance from the tetO array (one instance per strain). Selection for HIS3 (diamonds) decreases the mutation rate slightly and addition of dox (squares) eliminates targeted mutagenesis completely. (D) TaGTEAM also functions when targeted to a plasmid containing the tetO array. Mutation rates were monitored using the gain of function marker ade2-1, which reverts through base pair substitutions at an internal stop codon. Labels on data points report the ability to PCR KlURA3 from a particular mutant, PCR+(total). Error bars are 95% CI.
Mentions: We chose to use a DNA glycosylase as our mutator enzyme and localize it by fusion with the tet repressor (tetR) that binds the 19-bp tet operator (tetO) sequence. DNA glycosylases normally function as the first step in base excision repair (BER) to remove chemically altered DNA bases. A build-up of unprocessed abasic sites leads to replication fork stalling and recruitment of error-prone trans-lesion polymerases (26). This faulty repair can lead to both point mutations and frameshifts (27). We tested the yeast 3-methyladenine glycosylase Mag1p, which is primarily responsible for excising alkylated bases, but has naturally broad substrate specificity (28) and is thought to excise normal base pairs when overexpressed (29). We fused Mag1p to single-chain (sc) tetR. Normal tetR forms a dimer; therefore, fusion to tetR may result in impaired enzymatic or binding activity because of a loss of conformational freedom caused by having two copies of Mag1p in such close proximity. Fusion of Mag1 to sctetR—a tandem repeat of tetR connected via a peptide linker (30)—results in one copy of Mag1 per tetR dimer and eliminates this problem (see Figure 1A). We then verified the mutator activity of Mag1-sctetR by overexpressing it in an apn1Δ background, as reduced AP (apurinic/apyrimidinic) endonuclease activity elevates mutation rates (29) (Supplementary Figure S1). Finally, we introduced a tetR-repressible promoter driving yeast fluorescent protein (YFP) (31) in cells expressing Mag1-sctetR to confirm it bound tetO sites. When doxycycline (dox) is added, it binds to and reduces the affinity of sctetR for tetO, relieving the repression and increasing YFP expression. Mag1-sctetR repressed YFP expression to nearly the same extent as sctetR (Supplementary Figure S1E). Therefore, Mag1-sctetR has both mutagenic and specific DNA-binding activity. Similar experiments using cytosine DNA glycosylase (CDG), a mutant of human uracil DNA glycosylase specific for excising cytosines (32), did not yield targeted mutagenesis, probably because of the low expression of the CDG–sctetR fusion (Supplementary Figures S1 and S2).Figure 1.

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