PDBsum entry: 2atq

Transferase/DNA binding protein

PDB id: 2atq

Contents

Protein chains

845 a.a. *

210 a.a. *

Ligands

GDP

Metals

_ZN

* Residue conservation analysis

PDB id:

2atq

Name:

Transferase/DNA binding protein

Title:

Rb69 single-stranded DNA binding protein-DNA polymerase fusion

Structure:

DNA polymerase. Chain: a. Synonym: gp43. Engineered: yes. Mutation: yes. Other_details: c-terminus is fused to a linker not seen in the density. Gp32. Chain: b.

Source:

Enterobacteria phage rb69. Organism_taxid: 12353. Gene: 43. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008. Gene: gp32.

Resolution:

3.20Å R-factor: 0.284 R-free: 0.345

Authors:

S.Sun,L.Geng,Y.Shamoo

Key ref:

S.Sun et al. (2006). Structure and enzymatic properties of a chimeric bacteriophage RB69 DNA polymerase and single-stranded DNA binding protein with increased processivity. Proteins, 65, 231-238. PubMed id: 16881051 DOI: 10.1002/prot.21088

Date:

25-Aug-05 Release date: 09-May-06

Protein chain

Q38087  (DPOL_BPR69) -  DNA polymerase

Seq: 903 a.a.

Struc: 845 a.a.*

Protein chain

Q7Y265  (Q7Y265_BPR69) -  Gp32

Seq: 299 a.a.

Struc: 210 a.a.

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

Enzyme reactions

• Enzyme class: Chain A: E.C.2.7.7.7 - DNA-directed Dna polymerase.
• Reaction: Deoxynucleoside triphosphate + DNA(n) = diphosphate + DNA(n+1)

Deoxynucleoside triphosphate (Bound ligand (Het Group name = GDP) matches with 48.00% similarity) + DNA(n) = diphosphate + DNA(n+1)

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

 Gene Ontology (GO) functional annotation 

• Biological process: nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (2 terms)
• Biochemical function: nucleotide binding (10 terms)

reference

DOI no: 10.1002/prot.21088

Proteins 65:231-238 (2006)

PubMed id: 16881051

Structure and enzymatic properties of a chimeric bacteriophage RB69 DNA polymerase and single-stranded DNA binding protein with increased processivity.

S.Sun, L.Geng, Y.Shamoo.

ABSTRACT

In vivo, replicative DNA polymerases are made more processive by their interactions with accessory proteins at the replication fork. Single-stranded DNA binding protein (SSB) is an essential protein that binds tightly and cooperatively to single-stranded DNA during replication to remove adventitious secondary structures and protect the exposed DNA from endogenous nucleases. Using information from high resolution structures and biochemical data, we have engineered a functional chimeric enzyme of the bacteriophage RB69 DNA polymerase and SSB with substantially increased processivity. Fusion of RB69 DNA polymerase with its cognate SSB via a short six amino acid linker increases affinity for primer-template DNA by sixfold and subsequently increases processivity by sevenfold while maintaining fidelity. The crystal structure of this fusion protein was solved by a combination of multiwavelength anomalous diffraction and molecular replacement to 3.2 A resolution and shows that RB69 SSB is positioned proximal to the N-terminal domain of RB69 DNA polymerase near the template strand channel. The structural and biochemical data suggest that SSB interactions with DNA polymerase are transient and flexible, consistent with models of a dynamic replisome during elongation.

Selected figure(s)

Figure 1.
Figure 1. F6 displays higher processivity than does RB69 DNA polymerase. Primers were 5 end labeled ^32P 20-mers and are complementary to different regions on M13mp18. Proteins were incubated for 2 min at 30°C with annealed primer-template DNA and dNTPs. Reactions were initiated by the addition of MgCl[2] and stopped by the addition of EDTA/formamide loading buffer. Some of the reactions also have 500 g/mL heparin to produce single turn-over kinetics. Reaction products are separated on a 6% polyacrylamide-7M urea gel. Lane 1: Reaction mixture without enzyme. Lane 2: Heparin was added before F6 as a control to show that the fusion binds heparin and is an effective single-turnover sink. Lane 3-15: Reactions with different protein and protein combinations with/without heparin at different time points showing that compared with RB69 DNA polymerase, F6 has increased processivity. A. Reactions with P1 as primer. B. The primer P2 is complementary to a region on M13mp18, where there is a major replication pause site (12 bp hairpin structure) 105 nt downstream. As shown in Lane 3, F6 was able to overcome this major pause site and continue DNA synthesis, while all other protein and protein combinations stopped.

Figure 5.
Figure 5. F6 structure. A. Composite omit map showing part of the density for DNA polymerase in F6 at 1.5 level. B. Ribbon diagram of F6 structure with a zinc atom bound by SSB and a GDP bound by DNA polymerase. Color coding is based on crystallographic temperature factors (B), with red representing regions of higher B factors and blue representing regions of lower B factors (B factor range 1-100). The linker and 12 amino acids from the C-terminus of RB69 SSB core domain are highly flexible and were not seen in the electron density region connecting the C-terminus of SSB to N-terminus of DNA polymerase. C. Surface charge distribution of F6.

The above figures are reprinted by permission from John Wiley & Sons, Inc.: Proteins (2006, 65, 231-238) copyright 2006.

Figures were selected by the author.