PDBsum entry 4z20

Hydrolase/DNA

PDB id: 4z20

Contents

Protein chains

298 a.a.

DNA/RNA

Ligands

GOL ×3

Metals

_CA ×2

Waters ×11

PDB id:

4z20

Name:

Hydrolase/DNA

Title:

Crystal structure of meganuclease i-smami bound to uncleaveable DNA with a ttgt central four

Structure:

Meganuclease i-smami. Chain: a, d. Engineered: yes. DNA (26-mer). Chain: c, f. Engineered: yes. DNA (26-mer). Chain: b, e. Engineered: yes

Source:

Sordaria macrospora (strain atcc mya-333 / dsm 997 / k(l3346) / k-hell). Organism_taxid: 771870. Strain: atcc mya-333 / dsm 997 / k(l3346) / k-hell. Gene: smac_12671. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Synthetic construct.

Resolution:

3.20Å R-factor: 0.236 R-free: 0.291

Authors:

J.P.Hallinan,B.L.Stoddard

Key ref:

A.R.Lambert et al. (2016). Indirect DNA Sequence Recognition and Its Impact on Nuclease Cleavage Activity. Structure, 24, 862-873. PubMed id: 27133026 DOI: 10.1016/j.str.2016.03.024

Date:

27-Mar-15 Release date: 30-Mar-16

Protein chains

F7WD42  (F7WD42_SORMK) -  Homing endonuclease LAGLIDADG domain-containing protein from Sordaria macrospora (strain ATCC MYA-333 / DSM 997 / K(L3346) / K-hell)

Seq: 432 a.a.

Struc: 298 a.a.*

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

DNA/RNA chains

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

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

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

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

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1016/j.str.2016.03.024

Structure 24:862-873 (2016)

PubMed id: 27133026

Indirect DNA Sequence Recognition and Its Impact on Nuclease Cleavage Activity.

A.R.Lambert, J.P.Hallinan, B.W.Shen, J.K.Chik, J.M.Bolduc, N.Kulshina, L.I.Robins, B.K.Kaiser, J.Jarjour, K.Havens, A.M.Scharenberg, B.L.Stoddard.

ABSTRACT

LAGLIDADG meganucleases are DNA cleaving enzymes used for genome engineering. While their cleavage specificity can be altered using several protein engineering and selection strategies, their overall targetability is limited by highly specific indirect recognition of the central four base pairs within their recognition sites. In order to examine the physical basis of indirect sequence recognition and to expand the number of such nucleases available for genome engineering, we have determined the target sites, DNA-bound structures, and central four cleavage fidelities of nine related enzymes. Subsequent crystallographic analyses of a meganuclease bound to two noncleavable target sites, each containing a single inactivating base pair substitution at its center, indicates that a localized slip of the mutated base pair causes a small change in the DNA backbone conformation that results in a loss of metal occupancy at one binding site, eliminating cleavage activity.