PDBsum entry 1xns

Hydrolase, ligase/DNA

PDB id: 1xns

Contents

Protein chains

322 a.a. *

DNA/RNA

Waters ×296

* Residue conservation analysis

PDB id:

1xns

Name:

Hydrolase, ligase/DNA

Title:

Peptide trapped holliday junction intermediate in cre-loxp recombination

Structure:

Loxp DNA. Chain: c. Engineered: yes. Other_details: part of holliday junction. Loxp DNA. Chain: d. Engineered: yes. Other_details: part of holliday junction. Recombinase cre.

Source:

Synthetic: yes. Enterobacteria phage p1. Organism_taxid: 10678. Gene: cre. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Biol. unit:

Octamer (from PDB file)

Resolution:

2.80Å R-factor: 0.200 R-free: 0.265

Authors:

K.Ghosh,C.K.Lau,F.Guo,A.M.Segall,G.D.Van Duyne

Key ref:

K.Ghosh et al. (2005). Peptide trapping of the Holliday junction intermediate in Cre-loxP site-specific recombination. J Biol Chem, 280, 8290-8299. PubMed id: 15591069 DOI: 10.1074/jbc.M411668200

Date:

05-Oct-04 Release date: 14-Dec-04

Protein chains

P06956  (RECR_BPP1) -  Recombinase cre from Escherichia phage P1

Seq: 343 a.a.

Struc: 322 a.a.

DNA/RNA chains

T-A-T-A-A-C-T-T-C-G-T-A-T-A-A-T-G-T-A-T-G-C-T-A-T-A-C-G-A-A-G-T-T-A-T 35 bases

A-T-A-A-C-T-T-C-G-T-A-T-A-G-C-A-T-A-C-A-T-T-A-T-A-C-G-A-A-G-T-T-A-T 34 bases

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1074/jbc.M411668200

J Biol Chem 280:8290-8299 (2005)

PubMed id: 15591069

Peptide trapping of the Holliday junction intermediate in Cre-loxP site-specific recombination.

K.Ghosh, C.K.Lau, F.Guo, A.M.Segall, G.D.Van Duyne.

ABSTRACT

Cre recombinase is a prototypical member of the tyrosine recombinase family of site-specific recombinases. Members of this family of enzymes catalyze recombination between specific DNA sequences by cleaving and exchanging one pair of strands between the two substrate sites to form a 4-way Holliday junction (HJ) intermediate and then resolve the HJ intermediate to recombinant products by a second round of strand exchanges. Recently, hexapeptide inhibitors have been described that are capable of blocking the second strand exchange step in the tyrosine recombinase recombination pathway, leading to an accumulation of the HJ intermediate. These peptides are active in the lambda-integrase, Cre recombinase, and Flp recombinase systems and are potentially important tools for both in vitro mechanistic studies and as in vivo probes of cellular function. Here we present biochemical and crystallographic data that support a model where the peptide inhibitor binds in the center of the recombinase-bound DNA junction and interacts with solvent-exposed bases near the junction branch point. Peptide binding induces large conformational changes in the DNA strands of the HJ intermediate, which affect the active site geometries in the recombinase subunits.

Selected figure(s)

Figure 4.
FIG. 4. Difference electron density at 2.8-Å resolution in the center of the Cre-loxPHJ-peptide complex. The density is contoured at 2.3 times the r.m.s. value of the map. Corresponding density is not observed in the Cre-HJ1, Cre-HJ2, or Cre-loxPHJ structures that were crystallized in the absence of peptide inhibitor under similar conditions (Table II). Density regions marked A and B are discussed in the text. Cre recombinase subunits are not shown. This figure was created with Pymol (43).

Figure 6.
FIG. 6. Comparison of active site geometries between Cre-loxPHJ-peptide (in gold) and Cre-loxPHJ (in red) intermediates. A, stereo view of a superposition of the cleaving active site that normally catalyzes resolution of this junction isomer to duplex products. B, stereo view of a superposition of the non-cleaving active site. The scissile phosphates are indicated by gold or red spheres. This figure was generated using Molscript (44).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2005, 280, 8290-8299) copyright 2005.

Figures were selected by an automated process.