PDBsum entry 3ebc

Hydrolase/DNA

PDB id: 3ebc

Contents

Protein chains

241 a.a.

DNA/RNA

Metals

_MN

Waters ×301

PDB id:

3ebc

Name:

Hydrolase/DNA

Title:

Structure of n141a hincii with cognate DNA

Structure:

Type-2 restriction enzyme hincii. Chain: a, b. Synonym: r.Hincii, type ii restriction enzyme hincii, endonuclease hincii. Engineered: yes. Mutation: yes. 5'-d( Dgp Dcp Dcp Dgp Dgp Dtp Dcp Dgp Dap Dcp Dgp Dgp Dgp D c)-3'. Chain: e.

Source:

Haemophilus influenzae. Organism_taxid: 727. Gene: hinciir. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Synthetic: yes

Resolution:

2.55Å R-factor: 0.195 R-free: 0.279

Authors:

E.J.Little,A.C.Babic,N.C.Horton

Key ref:

E.J.Little et al. (2008). Early interrogation and recognition of DNA sequence by indirect readout. Structure, 16, 1828-1837. PubMed id: 19081059 DOI: 10.1016/j.str.2008.09.009

Date:

27-Aug-08 Release date: 23-Dec-08

Protein chains

P17743  (T2C2_HAEIF) -  Type II restriction enzyme HincII from Haemophilus influenzae

Seq: 258 a.a.

Struc: 241 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

DNA/RNA chains

G-C-C-G-G-T-C-G-A-C-G-G-G-C 14 bases

G-C-C-C-G-T-C-G-A-C-C-G-G-C 14 bases

Enzyme reactions

• Enzyme class: E.C.3.1.21.4 - type Ii site-specific deoxyribonuclease.
• Reaction: Endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates.
• Cofactor: Mg(2+)

DOI no: 10.1016/j.str.2008.09.009

Structure 16:1828-1837 (2008)

PubMed id: 19081059

Early interrogation and recognition of DNA sequence by indirect readout.

E.J.Little, A.C.Babic, N.C.Horton.

ABSTRACT

Control of replication, transcription, recombination and repair requires proteins capable of finding particular DNA sequences in a background of a large excess of nonspecific sequences. Such recognition can involve direct readout, with direct contacts to the bases of DNA, or in some cases through the less well-characterized indirect readout mechanisms. In order to measure the relative contributions of direct and indirect readout by a sequence specific endonuclease, HincII, a mutant enzyme deficient in a direct contact, was characterized, and surprisingly showed no loss of sequence specificity. The three dimensional crystal structure shows the loss of most of the direct readout contacts to the DNA, possibly capturing an early stage in target site recognition using predominately indirect readout to prescreen sites before full sequence interrogation.

Selected figure(s)

Figure 1.
Figure 1. DNA Binding and Distortion by Wild-Type HincII
(A) Two views of the structure of wild-type HincII (two subunits shown as black or white ribbons) bound to cognate DNA (shown in space filling with the recognition site DNA in light brown and the center two base pairs of the YR step in cyan and magenta, respectively, with flanking DNA in dark brown).
(B) Cartoon depiction of B form DNA (left) with that of the center YR step of the HincII DNA recognition sequence bound to wild-type HincII (right), in which the DNA bases of the same strand are unstacked and the purine bases from opposing strands exhibit greater stacking surface area (dashed lines) forming the cross-strand purine stack (CSPS).
(C) Numbering of the HincII recognition sequence used throughout the text.
(D) Hydrogen bonds between the side chain of N141 in wild-type HincII and bound cognate DNA.

Figure 4.
Figure 4. Protein DNA Contacts in Wild-Type and N141A HincII/DNA Structures
(A) Stereo view of the contacts to the DNA by residues 138–141 of N141A HincII. Colored by atom type: N-blue, O-red, P-orange, C-green in protein, pink in DNA of the recognition site and yellow in flanking base pairs. A water molecule is shown as a red sphere. Dashes indicate hydrogen bonds.
(B) Stereo view of the contacts to the DNA by residues 138–141 of wild-type HincII. Colored as in (A). Dashes indicate hydrogen bonds.
(C) Stereo view of the contacts to the DNA at Thy 2′ and Gua 1′ of N141A HincII. Colored as in (A). Methyl groups are shown as green or pink spheres; water molecule is shown as a red sphere. Dashes indicate hydrogen bonds or van der Waals interactions.
(D) Stereo view of the contacts to the DNA at Thy 2′ and Gua 1′ of wild-type HincII. Colored as in (A). Methyl groups are shown as green or pink spheres. Dashes indicate hydrogen bonds or van der Waals interactions.
(E) Cartoon of protein-DNA contacts to bases in the major groove in the N141A HincII/DNA structure. Water molecule is shown as a red circle.
(F) Cartoon of protein-DNA contacts to bases in the major groove in the wild-type HincII/DNA structure. Dashes indicate hydrogen bonds or van der Waals interactions.

The above figures are reprinted from an Open Access publication published by Cell Press: Structure (2008, 16, 1828-1837) copyright 2008.

Figures were selected by an automated process.