PDBsum entry 1u0c

Hydrolase/DNA

PDB id: 1u0c

Contents

Protein chains

152 a.a. *

DNA/RNA

Metals

_MG ×3

Waters ×90

* Residue conservation analysis

PDB id:

1u0c

Name:

Hydrolase/DNA

Title:

Y33c mutant of homing endonuclease i-crei

Structure:

5'-d( Gp Cp Tp Ap Ap Ap Cp Gp Tp Cp Gp Tp Gp Ap Gp Ap Cp Ap Gp Tp Tp Ap Cp G)-3'. Chain: c. Engineered: yes. 5'-d( Cp Gp Tp Ap Ap Cp Tp Gp Tp Cp Tp Cp Ap Cp Gp Ap Cp Gp Tp Tp Tp Ap Gp C)-3'. Chain: d. Engineered: yes. DNA endonuclease i-crei.

Source:

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

Biol. unit:

Tetramer (from PQS)

Resolution:

2.50Å R-factor: 0.214 R-free: 0.277

Ensemble:

2 models

Authors:

D.Sussman,M.Chadsey,S.Fauce,A.Engel,A.Bruett,R.Monnat,B.L.Stoddard, L.M.Seligman

Key ref:

D.Sussman et al. (2004). Isolation and characterization of new homing endonuclease specificities at individual target site positions. J Mol Biol, 342, 31-41. PubMed id: 15313605 DOI: 10.1016/j.jmb.2004.07.031

Date:

13-Jul-04 Release date: 02-Nov-04

Protein chains

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

Seq: 163 a.a.

Struc: 152 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

DNA/RNA chains

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

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

Enzyme reactions

• Enzyme class: E.C.3.1.-.-

DOI no: 10.1016/j.jmb.2004.07.031

J Mol Biol 342:31-41 (2004)

PubMed id: 15313605

Isolation and characterization of new homing endonuclease specificities at individual target site positions.

D.Sussman, M.Chadsey, S.Fauce, A.Engel, A.Bruett, R.Monnat, B.L.Stoddard, L.M.Seligman.

ABSTRACT

Homing endonucleases are highly specific DNA endonucleases, encoded within mobile introns or inteins, that induce targeted recombination, double-strand repair and gene conversion of their cognate target sites. Due to their biological function and high level of target specificity, these enzymes are under intense investigation as tools for gene targeting. These studies require that naturally occurring enzymes be redesigned to recognize novel target sites. Here, we report studies in which the homodimeric LAGLIDADG homing endonuclease I-CreI is altered at individual side-chains corresponding to contact points to distinct base-pairs in its target site. The resulting enzyme constructs drive specific elimination of selected DNA targets in vivo and display shifted specificities of DNA binding and cleavage in vitro. Crystal structures of two of these constructs demonstrate that substitution of individual side-chain/DNA contact patterns can occur with almost no structural deformation or rearrangement of the surrounding complex, facilitating an isolated, modular redesign strategy for homing endonuclease activity and specificity.

Selected figure(s)

Figure 1.
Figure 1. Structure of I-CreI and DNA target sites used in this study. (a) Structure of wild-type I-CreI bound to DNA. The positions of residues that are targeted for selection are indicated in the homodimer by red labels and arrows. (b) Wild-type and mutant enzyme DNA-binding sites. Base-pairs ±6 and ±10, that interact with Q26/Y66 and Y33, respectively, are colored to correspond to the scheme in the top panel. Points of cleavage are noted with blue triangles and red cleavage patterns.

Figure 4.
Figure 4. Structures and interactions of cognate pairs at residue 33 and base-pair 10. Protein-DNA contacts in the vicinity of base-pair ±10, in bound complexes containing either wild-type enzyme and DNA target site (left), or Y33C and Y33H mutant enzymes bound to their cognate target site (middle and right, respectively). The sequence of wild-type I-CreI target sequence (left) and alternate target sequences (middle and right) targeted for selection are shown below their corresponding structures.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2004, 342, 31-41) copyright 2004.

Figures were selected by an automated process.