Article

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

ACS Chemical Biology, American Chemical Society (ACS), ISSN 1554-8937

Volume 11, 5, 2016

DOI:10.1021/acschembio.5b00730, Dimensions: pub.1055135049, PMID: 26909878,

Affiliations

Organisations

  1. (1) Spanish National Cancer Research Centre, grid.7719.8
  2. (2) Cineca, grid.431603.3
  3. (3) University of Copenhagen, grid.5254.6, KU
  4. (4) Sapienza University of Rome, grid.7841.a

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Spain

Italy

Denmark

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Europe

Description

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.

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NORA University Profiles

University of Copenhagen

Danish Open Access Indicator

2016: Blocked

Research area: Medicine

Danish Bibliometrics Indicator

2016: Level 1

Research area: Medicine

Dimensions Citation Indicators

Times Cited: 3

Field Citation Ratio (FCR): 0.48

Relative Citation ratio (RCR): 0.29