Key Players in I-DmoI Endonuclease Catalysis Revealed from Structure and Dynamics

Rafael Molina; Neva Besker; Maria Jose Marcaida; Guillermo Montoya; Jesús Prieto; Marco D'Abramo

doi:10.1021/acschembio.5b00730

Key Players in I-DmoI Endonuclease Catalysis Revealed from Structure and Dynamics

Rafael Molina, Neva Besker, Maria Jose Marcaida, Guillermo Montoya, Jesús Prieto, Marco D'Abramo

Protein Structure and Function Program

5 Citationer (Scopus)

Abstract

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.

Originalsprog	Engelsk
Tidsskrift	ACS chemical biology
Vol/bind	11
Udgave nummer	5
Sider (fra-til)	1401–1407
ISSN	1554-8929
DOI	https://doi.org/10.1021/acschembio.5b00730
Status	Udgivet - 20 maj 2016

Adgang til dokumentet

10.1021/acschembio.5b00730

Citationsformater

@article{9cbc2e29a81f47968ca7b10a293d7b3e,

title = "Key Players in I-DmoI Endonuclease Catalysis Revealed from Structure and Dynamics",

abstract = "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 {\AA} 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.",

author = "Rafael Molina and Neva Besker and Marcaida, {Maria Jose} and Guillermo Montoya and Jes{\'u}s Prieto and Marco D'Abramo",

year = "2016",

month = may,

day = "20",

doi = "10.1021/acschembio.5b00730",

language = "English",

volume = "11",

pages = "1401–1407",

journal = "ACS chemical biology",

issn = "1554-8929",

publisher = "American Chemical Society",

number = "5",

}

TY - JOUR

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 - 2016/5/20

Y1 - 2016/5/20

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

SN - 1554-8929

VL - 11

SP - 1401

EP - 1407

JO - ACS chemical biology

JF - ACS chemical biology

IS - 5

ER -

Key Players in I-DmoI Endonuclease Catalysis Revealed from Structure and Dynamics

Abstract

Adgang til dokumentet

Fingeraftryk

Citationsformater