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

J.A.C.S. (2006), 128: 2477-84.

Library size: ~1×106

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 .

Protein Linker Sequence

mCreI TGSGSGSKSQAVAHPTDGQRDFGAKGSGSGSGT

mMsoI TGSGSGSKHPTLTLPTTTSQENLPNGSGSGSGT

Biochemical Characterization by Circular Dichroism (CD)

Protein Tm (ºC)

CreWT 66.81±0.01

mCre 56.46±0.07

• CreWT and mCre show distinctiv

e secondary structures and thermos

tability profile by far-UV CD.

Biochemical Characterization by Circular Dichroism (CD)

Protein Tm (ºC)

MsoWT 54.30±0.01

mMso 56.23±0.08

• MsoWT and mMso show si

milar secondary structures and

thermostability profile.

Thermodynamic Values of mCre by ITC

ProteinKD

nM

∆H

kcal/mol

∆S

cal/mol/deg

-T∆S

kcal/mol

∆G

kcal/mol

CreWT 13.3±2.6 19.6±0.20 101 -30.6 -11

mCre 25.8±4.7 26.7±0.59 123±1.0 -37.3 -11.5

CreWT mCre

Thermodynamic Values of mMso by ITC

mMso

ProteinKD

nM

∆H

kcal/mol

∆S

cal/mol/deg

-T∆S

kcal/mol

∆G

kcal/mol

MsoWT1 21±5.0 12.7±0.27 77±1.1 -23.3 -10.6

Mso 40.2±9.6 14.7±0.4 82.4 -25.0 -10.3

1. Eastberg et. al. Nucleic Acid Res. (2007), 35(21): 7209-21

MsoWT1

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

Competitive Binding Matrix of MsoWT and mMso

MsoWT

Lindsey Doyle

mMso

• MsoWT and mMso show similar binding specifities.

Site Specificity by Ultra-deep Sequencing

Solexa sequencing

probabilistic site models/ better PSSMs

Argast et. al. J Mol Biol. (1998), 280: 345-353

Sequential Enrichment of Cleavage-Sensitive Target Sites

• In each round, 1 pmol plasmid DNA was digested at 37ºC for 1 hour in presenc

e of 20 pmol enzyme.

• After five rounds of enrichment, the majority of library are cleavage-sensitive.

Summary

• Two monomeric I-CreI (mCre) and I-MsoI (mMso) were selected, and show compar

able 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 c

leavage 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 m

atrix) 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.