PDBsum entry 6bcf

Hydrolase/DNA

PDB id: 6bcf

Contents

Protein chains

288 a.a.

DNA/RNA

Metals

_CA ×12

PDB id:

6bcf

Name:

Hydrolase/DNA

Title:

I-ltri g183a bound to cognate substrate (pre-cleavage complex)

Structure:

Ribosomal protein 3/homing endonuclease-like fusion protein. Chain: d, a, g, j. Engineered: yes. DNA (26-mer). Chain: e, b, h, k. Engineered: yes. DNA (26-mer). Chain: f, c, i, l.

Source:

Leptographium truncatum. Organism_taxid: 330483. Gene: heg fusion, rps3. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Organism_taxid: 330483

Resolution:

2.92Å R-factor: 0.243 R-free: 0.309

Authors:

C.Brown,K.Zhang,T.A.Mcmurrough,G.B.Gloor,D.R.Edgell,M.Junop

Key ref:

T.A.McMurrough et al. (2018). Active site residue identity regulates cleavage preference of LAGLIDADG homing endonucleases. Nucleic Acids Res, 46, 11990-12007. PubMed id: 30357419 DOI: 10.1093/nar/gky976

Date:

20-Oct-17 Release date: 24-Oct-18

Protein chains

C7SWF3  (C7SWF3_9PEZI) -  Ribosomal protein 3/homing endonuclease-like fusion protein from Leptographium truncatum

Seq: 712 a.a.

Struc: 288 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DNA/RNA chains

G-G-T-C-T-A-A-A-C-G-T-C-G-T-A-T-A-G-G-A-G-C-A-T-T-T 26 bases

C-A-A-A-T-G-C-T-C-C-T-A-T-A-C-G-A-C-G-T-T-T-A-G-A-C 26 bases

G-G-T-C-T-A-A-A-C-G-T-C-G-T-A-T-A-G-G-A-G-C-A-T-T-T 26 bases

C-A-A-A-T-G-C-T-C-C-T-A-T-A-C-G-A-C-G-T-T-T-A-G-A-C 26 bases

G-G-T-C-T-A-A-A-C-G-T-C-G-T-A-T-A-G-G-A-G-C-A-T-T-T 26 bases

C-A-A-A-T-G-C-T-C-C-T-A-T-A-C-G-A-C-G-T-T-T-A-G-A-C 26 bases

G-G-T-C-T-A-A-A-C-G-T-C-G-T-A-T-A-G-G-A-G-C-A-T-T-T 26 bases

C-A-A-A-T-G-C-T-C-C-T-A-T-A-C-G-A-C-G-T-T-T-A-G-A-C 26 bases

DOI no: 10.1093/nar/gky976

Nucleic Acids Res 46:11990-12007 (2018)

PubMed id: 30357419

Active site residue identity regulates cleavage preference of LAGLIDADG homing endonucleases.

T.A.McMurrough, C.M.Brown, K.Zhang, G.Hausner, M.S.Junop, G.B.Gloor, D.R.Edgell.

ABSTRACT

LAGLIDADG homing endonucleases (meganucleases) are site-specific mobile endonucleases that can be adapted for genome-editing applications. However, one problem when reprogramming meganucleases on non-native substrates is indirect readout of DNA shape and flexibility at the central 4 bases where cleavage occurs. To understand how the meganuclease active site regulates DNA cleavage, we used functional selections and deep sequencing to profile the fitness landscape of 1600 I-LtrI and I-OnuI active site variants individually challenged with 67 substrates with central 4 base substitutions. The wild-type active site was not optimal for cleavage on many substrates, including the native I-LtrI and I-OnuI targets. Novel combinations of active site residues not observed in known meganucleases supported activity on substrates poorly cleaved by the wild-type enzymes. Strikingly, combinations of E or D substitutions in the two metal-binding residues greatly influenced cleavage activity, and E184D variants had a broadened cleavage profile. Analyses of I-LtrI E184D and the wild-type proteins co-crystallized with the non-cognate AACC central 4 sequence revealed structural differences that correlated with kinetic constants for cleavage of individual DNA strands. Optimizing meganuclease active sites to enhance cleavage of non-native central 4 target sites is a straightforward addition to engineering workflows that will expand genome-editing applications.