PDBsum entry 1j5f

Hydrolase

PDB id: 1j5f

Contents

Protein chain

270 a.a.*

DNA/RNA

Metals

_MN ×2

* C-alpha coords only

Theoretical model

PDB id:

1j5f

Name:

Hydrolase

Title:

Revised model of t5 5' nuclease + DNA

Structure:

Exodeoxyribonuclease. Chain: a. Synonym: t5 5' nuclease, 5'exonuclease. 5'-d(p Ap Tp Gp Gp Cp Ap Ap Ap Gp Tp Ap Ap T)-3'. Chain: y. Engineered: yes. 5'- d(p Cp Ap Tp Cp Tp Ap Cp Tp Ap Cp Tp Tp Tp Gp Cp Cp Ap T)- 3'.

Source:

Bacteriophage t5. Synthetic: yes. Synthetic: yes

Authors:

J.J.Dervan,M.Feng,J.A.Grasby,P.J.Artymiuk,T.A.Ceska, J.R.Sayers

Key ref:

J.J.Dervan et al. (2002). Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding. Proc Natl Acad Sci U S A, 99, 8542-8547. PubMed id: 12084915 DOI: 10.1073/pnas.082241699

Date:

25-Apr-02 Release date: 22-May-02

Protein chain

P06229  (EXO5_BPT5) -  Exodeoxyribonuclease

Seq: 291 a.a.

Struc: 270 a.a.

DNA/RNA chains

A-T-G-G-C-A-A-A-G-T-A-A-T 13 bases

C-A-T-C-T-A-C-T-A-C-T-T-T-G-C-C-A-T 18 bases

Enzyme reactions

• Enzyme class: E.C.3.1.11.3 - exodeoxyribonuclease (lambda-induced).
• Reaction: Degradation of double-stranded DNA. It acts progressively in a 5'- to 3'-direction, releasing nucleoside 5'-phosphates.

DOI no: 10.1073/pnas.082241699

Proc Natl Acad Sci U S A 99:8542-8547 (2002)

PubMed id: 12084915

Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding.

J.J.Dervan, M.Feng, D.Patel, J.A.Grasby, P.J.Artymiuk, T.A.Ceska, J.R.Sayers.

ABSTRACT

Previous structural studies on native T5 5' nuclease, a member of the flap endonuclease family of structure-specific nucleases, demonstrated that this enzyme possesses an unusual helical arch mounted on the enzyme's active site. Based on this structure, the protein's surface charge distribution, and biochemical analyses, a model of DNA binding was proposed in which single-stranded DNA threads through the archway. We investigated the kinetic and substrate-binding characteristics of wild-type and mutant nucleases in relation to the proposed model. Five basic residues R33, K215, K241, R172, and R216, are all implicated in binding branched DNA substrates. All these residues except R172 are involved in binding to duplex DNA carrying a 5' overhang. Replacement of either K215 or R216 with a neutral amino acid did not alter kcat appreciably. However, these mutant nucleases displayed significantly increased values for Kd and Km. A comparison of flap endonuclease binding to pseudoY substrates and duplexes with a single-stranded 5' overhang suggests a better model for 5' nuclease-DNA binding. We propose a major revision to the binding model consistent with these biophysical data.

Selected figure(s)

Figure 1.
Fig. 1. Diagrammatic representation of the flap, Ps-Y, 5OVH, and HP1 oligonucleotide substrates referred to in this study. For each substrate the 5'-32P end-labeled oligonucleotide is shown as 5'-P. Exonucleolytic and structure-specific endonucleolytic cleavage sites are identified by filled and open triangles, respectively. I and II indicate duplex regions I and II, respectively.

Figure 2.
Fig. 2. (A) The structure (PDB ID code 1EXN) determined for T5 5' nuclease showing the helical arch (red backbone), divalent metal ions (gray spheres), and space-filling representations of selected lysine (blue) and arginine (cyan) residues. (B) Original DNA-binding model proposed by Ceska et al. (8). The duplex parts (I and II) of the substrate lie across a slightly concave and positively charged region of the protein. In this model residues K241, K215, and R216 contact duplex I, and R172 contacts duplex II.

Figures were selected by an automated process.