PDBsum entry 6fb5

DNA binding protein

PDB id: 6fb5

Contents

Protein chains

153 a.a.

154 a.a.

DNA/RNA

Ligands

ACT

PGO

Metals

_MG ×4

Waters ×121

PDB id:

6fb5

Name:

DNA binding protein

Title:

Crystal structure of a tailored i-crei homing endonuclease protein (3115 variant) in complex with an altered version of its target DNA (haemoglobin beta subunit gene) at 5nnn region in the presence of magnesium

Structure:

I-crei monomer a. Chain: a. Engineered: yes. I-crei monomer b. Chain: b. Engineered: yes. DNA (5'- d( Tp Cp Ap Gp Ap Cp Tp Tp Gp Tp Cp Cp Ap Cp Ap Gp Gp Ap Gp Tp Cp Ap Gp A)-3').

Source:

Chlamydomonas reinhardtii. Organism_taxid: 3055. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Synthetic construct. Organism_taxid: 32630. Organism_taxid: 32630

Resolution:

2.20Å R-factor: 0.191 R-free: 0.219

Authors:

R.Molina,J.Prieto

Key ref:

J.Prieto et al. (2018). Understanding the indirect DNA read-out specificity of I-CreI Meganuclease. Sci Rep, 8, 10286. PubMed id: 29980759

Date:

18-Dec-17 Release date: 18-Jul-18

Protein chain

P05725  (DNE1_CHLRE) -  DNA endonuclease I-CreI from Chlamydomonas reinhardtii

Seq: 163 a.a.

Struc: 153 a.a.*

Protein chain

P05725  (DNE1_CHLRE) -  DNA endonuclease I-CreI from Chlamydomonas reinhardtii

Seq: 163 a.a.

Struc: 154 a.a.*

* PDB and UniProt seqs differ at 17 residue positions (black crosses)

DNA/RNA chains

T-C-A-G-A-C-T-T-G-T-C-C-A-C-A-G-G-A-G-T-C-A-G-A 24 bases

T-C-T-G-A-C-T-C-C-T-G-T-G-G-A-C-A-A-G-T-C-T-G-A 24 bases

Enzyme reactions

• Enzyme class: Chains A, B: E.C.3.1.-.-

Sci Rep 8:10286 (2018)

PubMed id: 29980759

Understanding the indirect DNA read-out specificity of I-CreI Meganuclease.

J.Prieto, P.Redondo, B.López-Méndez, M.D'Abramo, N.Merino, F.J.Blanco, P.Duchateau, G.Montoya, R.Molina.

ABSTRACT

The high DNA specificity of homing endonucleases makes them a powerful protein scaffold to engineer enzymes for genome manipulation. Understanding their molecular recognition of DNA is an important prerequisite to generate engineered enzymes able to cleave DNA in specific desired genome sites. Protein-DNA recognition studies have been mostly focused on specific direct contacts between amino acid side chains and bases to redesign the binding interface. However, the important role of indirect readout in the central region of the target DNA of the homing endonuclease I-CreI suggested that indirect readout may play a key role in the redesign of protein-DNA interactions. The sequences of the I-CreI central substrate region, 2NN, along with the adjacent 5NNN, are key for substrate cleavage. Here, we analyse the mechanism of target discrimination at the 5NNN region by the I-CreI protein, revealing its critical role in the location and occupancy of the catalytic metal ions, which is crucial for cleavage. Our data highlight the importance of indirect readout for target DNA cleavage, thus aiding I-CreI engineering when targeting new DNA sequences.