title: Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM.
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.
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Shawn P
authors.initials: SP
authors.lastname: Finney-Manchester
authors.forename: Narendra
authors.initials: N
authors.lastname: Maheshri
mesh_terms: D045647:DNA Glycosylases / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D059645:Mutation Rate; D009875:Operator Regions, Genetic; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D059767:Recombinational DNA Repair; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific; D045647:DNA Glycosylases; C445668:MAG1 protein, S cerevisiae