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Monomerization of Homing Endonucleases

Monomerization of Homing Endonucleases. Targets: CreI, MsoI, and CeuI. Goal: Generation of catalytically active monomeric HEs by connecting two copies of protein with a linker. Monomerization of Homing Endonucleases. Gene synthesis. Protein Science (2003), 12: 197-206.

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Monomerization of Homing Endonucleases

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  1. Monomerization of Homing Endonucleases • Targets: CreI, MsoI, and CeuI. • Goal: Generation of catalytically active monomeric HEs by connecting two • copies of protein with a linker.

  2. Monomerization of Homing Endonucleases Gene synthesis Protein Science (2003), 12: 197-206 Library size: ~1×106 J.A.C.S. (2006), 128: 2477-84.

  3. Monomeric Cre3 (mCre) and Mso24 (mMso) • With a 33 Aa linker between two HE domains,both mCre and mMso show comparable site-specific cleavage activity as WT HEs. • Size-exclusion chromatography confirms the monomeric state of both proteins .

  4. Biochemical Characterization by Circular Dichroism (CD) • CreWT and mCre show distinctive secondary structures and thermostability profile by far-UV CD.

  5. Biochemical Characterization by Circular Dichroism (CD) • MsoWT and mMso show similar secondary structures and thermostability profile.

  6. Thermodynamic Values of mCre by ITC CreWT mCre

  7. Thermodynamic Values of mMso by ITC MsoWT1 mMso 1. Eastberg et. al. Nucleic Acid Res. (2007), 35(21): 7209-21

  8. Competitive Binding Matrix KaA KaWT (FNC-FA)×FWT FA×(FNC-FWT) = Ka = association constant F = fluorescence A = oligos with one bp mismatch WT = WT oligos NC = random oligos Lei Zhao

  9. Competitive Binding Matrix of MsoWT and mMso MsoWT Lindsey Doyle mMso • MsoWT and mMso show similar binding specifities.

  10. Site Specificity by Ultra-deep Sequencing probabilistic site models/ better PSSMs Solexa sequencing Argast et. al. J Mol Biol. (1998), 280: 345-353

  11. Sequential Enrichment of Cleavage-Sensitive Target Sites • In each round, 1 pmol plasmid DNA was digested at 37ºC for 1 hour in presence of 20 pmol enzyme. • After five rounds of enrichment, the majority of library are cleavage-sensitive.

  12. Summary • Two monomeric I-CreI (mCre) and I-MsoI (mMso) were selected, and show comparable in vitro cleavage activity as WT enzymes. • mCre shows distinctive structural features and DNA binding. • mMso shows similar structural, biochemical characteristics as I-MsoI. Future Works • Crystallization of mCre and mMso. • Incorporate Mso mutations in silico designed by Justin into mMso, and validate the cleavage of asymmetric DNA target site by mMso mutants. • High-throughput sequencing and analysis of cleavage sensitive libraries by CreWT, MsoWT, and their monomeric version, and generate PSSM (position specific search matrix) for each individual enzyme. • Search for potential target sites in genes of interest using new PSSMs. • Engineering of Cre towards physiologically relevant target sites, and incorporate into monomeric version.

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