Key Players in I-DmoI Endonuclease Catalysis Revealed from Structure and Dynamics
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Key Players in I-DmoI Endonuclease Catalysis Revealed from Structure and Dynamics. / Molina, Rafael; Besker, Neva; Marcaida, Maria Jose; Montoya, Guillermo; Prieto, Jesús; D'Abramo, Marco.
In: ACS chemical biology, Vol. 11, No. 5, 08.03.2016, p. 1401–1407.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - Key Players in I-DmoI Endonuclease Catalysis Revealed from Structure and Dynamics
AU - Molina, Rafael
AU - Besker, Neva
AU - Marcaida, Maria Jose
AU - Montoya, Guillermo
AU - Prieto, Jesús
AU - D'Abramo, Marco
PY - 2016/3/8
Y1 - 2016/3/8
N2 - Homing endonucleases, such as I-DmoI, specifically recognize and cleave long DNA target sequences (∼20 bp) and are potentially powerful tools for genome manipulation. However, inefficient and off-target DNA cleavage seriously limits specific editing in complex genomes. One approach to overcome these limitations is to unambiguously identify the key structural players involved in catalysis. Here, we report the E117A I-DmoI mutant crystal structure at 2.2 Å resolution that, together with the wt and Q42A/K120M constructs, is combined with computational approaches to shed light on protein cleavage activity. The cleavage mechanism was related both to key structural effects, such as the position of water molecules and ions participating in the cleavage reaction, and to dynamical effects related to protein behavior. In particular, we found that the protein perturbation pattern significantly changes between cleaved and noncleaved DNA strands when the ions and water molecules are correctly positioned for the nucleophilic attack that initiates the cleavage reaction, in line with experimental enzymatic activity. The proposed approach paves the way for an effective, general, and reliable procedure to analyze the enzymatic activity of endonucleases from a very limited data set, i.e., structure and dynamics.
AB - Homing endonucleases, such as I-DmoI, specifically recognize and cleave long DNA target sequences (∼20 bp) and are potentially powerful tools for genome manipulation. However, inefficient and off-target DNA cleavage seriously limits specific editing in complex genomes. One approach to overcome these limitations is to unambiguously identify the key structural players involved in catalysis. Here, we report the E117A I-DmoI mutant crystal structure at 2.2 Å resolution that, together with the wt and Q42A/K120M constructs, is combined with computational approaches to shed light on protein cleavage activity. The cleavage mechanism was related both to key structural effects, such as the position of water molecules and ions participating in the cleavage reaction, and to dynamical effects related to protein behavior. In particular, we found that the protein perturbation pattern significantly changes between cleaved and noncleaved DNA strands when the ions and water molecules are correctly positioned for the nucleophilic attack that initiates the cleavage reaction, in line with experimental enzymatic activity. The proposed approach paves the way for an effective, general, and reliable procedure to analyze the enzymatic activity of endonucleases from a very limited data set, i.e., structure and dynamics.
U2 - 10.1021/acschembio.5b00730
DO - 10.1021/acschembio.5b00730
M3 - Journal article
C2 - 26909878
VL - 11
SP - 1401
EP - 1407
JO - A C S Chemical Biology
JF - A C S Chemical Biology
SN - 1554-8929
IS - 5
ER -
ID: 157550308