5'-Cytosine-phosphoguanine (CpG) methylation impacts the activity of natural and engineered meganucleases
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5'-Cytosine-phosphoguanine (CpG) methylation impacts the activity of natural and engineered meganucleases. / Valton, Julien; Daboussi, Fayza; Leduc, Sophie; Molina, Rafael; Redondo, Pilar; Macmaster, Rachel; Montoya, Guillermo; Duchateau, Philippe.
In: The Journal of Biological Chemistry, Vol. 287, No. 36, 31.08.2012, p. 30139-50.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - 5'-Cytosine-phosphoguanine (CpG) methylation impacts the activity of natural and engineered meganucleases
AU - Valton, Julien
AU - Daboussi, Fayza
AU - Leduc, Sophie
AU - Molina, Rafael
AU - Redondo, Pilar
AU - Macmaster, Rachel
AU - Montoya, Guillermo
AU - Duchateau, Philippe
PY - 2012/8/31
Y1 - 2012/8/31
N2 - In this study, we asked whether CpG methylation could influence the DNA binding affinity and activity of meganucleases used for genome engineering applications. A combination of biochemical and structural approaches enabled us to demonstrate that CpG methylation decreases I-CreI DNA binding affinity and inhibits its endonuclease activity in vitro. This inhibition depends on the position of the methylated cytosine within the DNA target and was almost total when it is located inside the central tetrabase. Crystal structures of I-CreI bound to methylated cognate target DNA suggested a molecular basis for such inhibition, although the precise mechanism still has to be specified. Finally, we demonstrated that the efficacy of engineered meganucleases can be diminished by CpG methylation of the targeted endogenous site, and we proposed a rational design of the meganuclease DNA binding domain to alleviate such an effect. We conclude that although activity and sequence specificity of engineered meganucleases are crucial parameters, target DNA epigenetic modifications need to be considered for successful gene editions.
AB - In this study, we asked whether CpG methylation could influence the DNA binding affinity and activity of meganucleases used for genome engineering applications. A combination of biochemical and structural approaches enabled us to demonstrate that CpG methylation decreases I-CreI DNA binding affinity and inhibits its endonuclease activity in vitro. This inhibition depends on the position of the methylated cytosine within the DNA target and was almost total when it is located inside the central tetrabase. Crystal structures of I-CreI bound to methylated cognate target DNA suggested a molecular basis for such inhibition, although the precise mechanism still has to be specified. Finally, we demonstrated that the efficacy of engineered meganucleases can be diminished by CpG methylation of the targeted endogenous site, and we proposed a rational design of the meganuclease DNA binding domain to alleviate such an effect. We conclude that although activity and sequence specificity of engineered meganucleases are crucial parameters, target DNA epigenetic modifications need to be considered for successful gene editions.
KW - CpG Islands
KW - Crystallography, X-Ray
KW - DNA/chemistry
KW - DNA Methylation
KW - DNA Restriction Enzymes/chemistry
KW - Epigenesis, Genetic
KW - HEK293 Cells
KW - Humans
KW - Protein Structure, Tertiary
U2 - 10.1074/jbc.M112.379966
DO - 10.1074/jbc.M112.379966
M3 - Journal article
C2 - 22740697
VL - 287
SP - 30139
EP - 30150
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 36
ER -
ID: 203018819