PDBsum entry 1b43

Transferase

PDB id: 1b43

Contents

Protein chains

339 a.a. *

Waters ×413

* Residue conservation analysis

PDB id:

1b43

Name:

Transferase

Title:

Fen-1 from p. Furiosus

Structure:

Protein (fen-1). Chain: a, b. Engineered: yes. Other_details: residue 1 in the structure is the first ordered residue, which corresponds to gly2 in the sequence.

Source:

Pyrococcus furiosus. Organism_taxid: 2261. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.00Å R-factor: 0.231 R-free: 0.279

Authors:

D.J.Hosfield,C.D.Mol,B.Shen,J.A.Tainer

Key ref:

D.J.Hosfield et al. (1998). Structure of the DNA repair and replication endonuclease and exonuclease FEN-1: coupling DNA and PCNA binding to FEN-1 activity. Cell, 95, 135-146. PubMed id: 9778254 DOI: 10.1016/S0092-8674(00)81789-4

Date:

05-Jan-99 Release date: 12-Jan-00

Protein chains

O93634  (FEN_PYRFU) -  Flap endonuclease 1 from Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)

Seq: 340 a.a.

Struc: 339 a.a.

Enzyme reactions

• Enzyme class: E.C.3.1.-.-

DOI no: 10.1016/S0092-8674(00)81789-4

Cell 95:135-146 (1998)

PubMed id: 9778254

Structure of the DNA repair and replication endonuclease and exonuclease FEN-1: coupling DNA and PCNA binding to FEN-1 activity.

D.J.Hosfield, C.D.Mol, B.Shen, J.A.Tainer.

ABSTRACT

Flap endonuclease (FEN-1) removes 5' overhanging flaps in DNA repair and processes the 5' ends of Okazaki fragments in lagging strand DNA synthesis. The crystal structure of Pyrococcus furiosus FEN-1, active-site metal ions, and mutational information indicate interactions for the single- and double-stranded portions of the flap DNA substrate and identify an unusual DNA-binding motif. The enzyme's active-site structure suggests that DNA binding induces FEN-1 to clamp onto the cleavage junction to form the productive complex. The conserved FEN-1 C terminus binds proliferating cell nuclear antigen (PCNA) and positions FEN-1 to act primarily as an exonuclease in DNA replication, in contrast to its endonuclease activity in DNA repair. FEN-1 mutations altering PCNA binding should reduce activity during replication, likely causing DNA repeat expansions as seen in some cancers and genetic diseases.

Selected figure(s)

Figure 4.
Figure 4. Conserved Overall Fold and Active Sites of pFEN-1 and Bacteriophage 5′ Exonucleases(A) Overall polypeptide fold of pFEN-1, T4 RNaseH, and T5 5′ exonuclease illustrating conserved structural features colored as in Figure 2B. Each enzyme contains a similar H3TH motif (magenta), which in T4 RNaseH contains an additional loop insertion, but contains different ssDNA-binding motifs (green), including the pFEN-1 helical clamp and T5 5′ exonuclease helical arch. The corresponding region in T4 RNaseH is not seen in the crystal structure.(B) Stereo view of the 2.0 Å resolution refined pFEN-1 model (yellow, carbon; red, oxygen; blue, nitrogen) and 2F[obs]-F[calc] electron density (contoured at 1.0 σ) showing the conserved residues at the pFEN-1 active site. Data collected from pFEN-1 crystals soaked with 2 mM PbCl[2] and 500 mM MnSO[4] reveal metal site M-1 (F[metal] − F[nat] difference electron density colored yellow and contoured at 15 σ) and metal site M-2 (F[metal] − F[nat] difference electron density colored green and contoured at 5 σ), respectively.(C) Stereo view of the superposition of the pFEN-1 active site and metal ions (yellow) with the active site and metal ions of T4 RNaseH (blue). Absolutely conserved pFEN-1 Tyr-237, from the H3TH motif, penetrates into the active site more deeply than does Tyr-86 from the T4 enzyme. Differences in the nature and positions of the conserved active-site residues in T4 RNaseH and FEN-1 likely influence the metal site positions and the distances between them in each enzyme.

Figure 5.
Figure 5. The pFEN-1 dsDNA-Binding SiteThe pFEN-1 fold and dsDNA-binding site viewed along the DNA-binding cleft. A flap DNA substrate was modeled into the cleft with the phosphodiester backbone poised to interact with the H3TH domain (magenta). The helical clamp (green) caps the active site and may serve to stabilize the bound substrate by interacting with ssDNA and/or dsDNA. During repair, the 5′ flap strand (white) passes over the active site and through the helical clamp.

The above figures are reprinted by permission from Cell Press: Cell (1998, 95, 135-146) copyright 1998.

Figures were selected by an automated process.