PDBsum entry 2dtu

Transferase/DNA

PDB id: 2dtu

Contents

Protein chains

896 a.a. *

DNA/RNA

Waters ×857

* Residue conservation analysis

PDB id:

2dtu

Name:

Transferase/DNA

Title:

Crystal structure of the beta hairpin loop deletion variant of rb69 gp43 in complex with DNA containing an abasic site analog

Structure:

5'-d( Cp Gp (3Dr) p Cp Tp Tp Ap Tp Gp Ap Cp Ap Gp Cp Cp Gp Cp G)-3'. Chain: e, g, i, k. Engineered: yes. 5'-d( Gp Cp Gp Gp Cp Tp Gp Tp Cp Ap Tp Ap Ap Gp A)-3'. Chain: f, h, j, l. Engineered: yes. DNA polymerase. Chain: a, b, c, d.

Source:

Synthetic: yes. Enterobacteria phage rb69. Organism_taxid: 12353. Gene: 43. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Biol. unit:

Trimer (from PQS)

Resolution:

2.37Å R-factor: 0.223 R-free: 0.268

Authors:

P.Aller,M.Hogg,W.Konigsberg,S.S.Wallace,S.Doublie

Key ref:

M.Hogg et al. (2007). Structural and biochemical investigation of the role in proofreading of a beta hairpin loop found in the exonuclease domain of a replicative DNA polymerase of the B family. J Biol Chem, 282, 1432-1444. PubMed id: 17098747 DOI: 10.1074/jbc.M605675200

Date:

15-Jul-06 Release date: 05-Dec-06

Protein chains

Q38087  (DPOL_BPR69) -  DNA-directed DNA polymerase from Escherichia phage RB69

Seq: 903 a.a.

Struc: 896 a.a.*

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

DNA/RNA chains

C-G-3DR-C-T-T-A-T-G-A-C-A-G-C-C-G-C-G 18 bases

G-C-G-G-C-T-G-T-C-A-T-A-A-G-A 15 bases

C-G-3DR-C-T-T-A-T-G-A-C-A-G-C-C-G-C-G 18 bases

G-C-G-G-C-T-G-T-C-A-T-A-A-G-A 15 bases

C-G-3DR-C-T-T-A-T-G-A-C-A-G-C-C-G-C-G 18 bases

G-C-G-G-C-T-G-T-C-A-T-A-A-G-A 15 bases

C-G-3DR-C-T-T-A-T-G-A-C-A-G-C-C-G-C-G 18 bases

G-C-G-G-C-T-G-T-C-A-T-A-A-G-A 15 bases

Enzyme reactions

• Enzyme class: E.C.2.7.7.7 - DNA-directed Dna polymerase.
• Reaction: DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1074/jbc.M605675200

J Biol Chem 282:1432-1444 (2007)

PubMed id: 17098747

Structural and biochemical investigation of the role in proofreading of a beta hairpin loop found in the exonuclease domain of a replicative DNA polymerase of the B family.

M.Hogg, P.Aller, W.Konigsberg, S.S.Wallace, S.Doublié.

ABSTRACT

Replicative DNA polymerases, as exemplified by the B family polymerases from bacteriophages T4 and RB69, not only replicate DNA but also have the ability to proofread misincorporated nucleotides. Because the two activities reside in separate protein domains, polymerases must employ a mechanism that allows for efficient switching of the primer strand between the two active sites to achieve fast and accurate replication. Prior mutational and structural studies suggested that a beta hairpin structure located in the exonuclease domain of family B polymerases might play an important role in active site switching in the event of a nucleotide misincorporation. We show that deleting the beta hairpin loop in RB69 gp43 affects neither polymerase nor exonuclease activities. Single binding event studies with mismatched primer termini, however, show that the beta hairpin plays a role in maintaining the stability of the polymerase/DNA interactions during the binding of the primer DNA in the exonuclease active site but not on the return of the corrected primer to the polymerase active site. In addition, the deletion variant showed a more stable incorporation of a nucleotide opposite an abasic site. Moreover, in the 2.4 A crystal structure of the beta hairpin deletion variant incorporating an A opposite a templating furan, all four molecules in the crystal asymmetric unit have DNA in the polymerase active site, despite the presence of DNA distortions because of the misincorporation, confirming that the primer strand is not stably bound within the exonuclease active site in the absence of the beta hairpin loop.

Selected figure(s)

Figure 1.
FIGURE 1. Switching of the hairpin loop in response to nucleotide incorporation opposite a lesion. A, the closed ternary complex of RB69 gp43 trapped with an incoming dTTP opposite a templating A (PDB code 1IG9 (17)). The 5'-end of the template DNA (gray) stacks against Trp-574 (gold), and the hairpin is in an up position. B, an open binary complex of RB69 gp43 after successful incorporation of an A opposite a furan (PDB code 1RV2, chain C (15)). In this structure the 5'-end of the template is sandwiched between Phe-359 (purple) and the hairpin, which is in a down position.

Figure 7.
FIGURE 7. In the crystal structure of the hairpin deletion variant, all four molecules have DNA in the polymerase active site. A and B, two of the four complexes found in the asymmetric unit of the previously solved furan-dAMP binary complex (PDB code 1RV2 (15)). One complex has DNA in the exonuclease active center (molecule B) (A); the other has DNA in the polymerase active site (molecule C) (B). C and D, the equivalent complexes from the structure of the -variant inserting an A opposite furan. In this structure none of the four complexes within the asymmetric unit has DNA in the exonuclease (exo) domain. The polymerase domains are colored red for the palm, green for the thumb, blue for the fingers, cyan for the exonuclease (with the hairpin in black), and orange for the N-terminal domain. The primer strand is shown in magenta and the template in dark blue. The tip of the hairpin loop was disordered in A.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 1432-1444) copyright 2007.

Figures were selected by the author.