PDBsum entry 1b1f

Transferase

PDB id: 1b1f

Contents

Protein chain

405 a.a.

DNA/RNA

Ligands

AVP

Theoretical model

PDB id:

1b1f

Name:

Transferase

Title:

Model of rb69 DNA polymerase with t7 DNA polymerase primer/ template

Structure:

DNA polymerase. Chain: r. DNA (5'- d(p Cp Cp Tp Tp Gp Gp Cp Ap Cp Tp Gp Gp C)-3'). Chain: t. Fragment: polymerase domain. Engineered: yes. DNA (5'-d(p Gp Cp Cp Ap Gp Tp Gp Cp Cp A)-3'). Chain: p.

Source:

Bacteriophage rb69. Synthetic: yes. Synthetic: yes

Authors:

D.M.Coen

Key ref:

L.Huang et al. (1999). The enzymological basis for resistance of herpesvirus DNA polymerase mutants to acyclovir: relationship to the structure of alpha-like DNA polymerases. Proc Natl Acad Sci U S A, 96, 447-452. PubMed id: 9892653 DOI: 10.1073/pnas.96.2.447

Date:

20-Nov-98 Release date: 20-Jan-99

Protein chain

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

Seq: 903 a.a.

Struc: 405 a.a.

DNA/RNA chains

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

G-C-C-A-G-T-G-C-C-A 10 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

DNA(n) (Bound ligand (Het Group name = AVP) matches with 5312.00% similarity corresponds exactly) + 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate

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

Added reference

DOI no: 10.1073/pnas.96.2.447

Proc Natl Acad Sci U S A 96:447-452 (1999)

PubMed id: 9892653

The enzymological basis for resistance of herpesvirus DNA polymerase mutants to acyclovir: relationship to the structure of alpha-like DNA polymerases.

L.Huang, K.K.Ishii, H.Zuccola, A.M.Gehring, C.B.Hwang, J.Hogle, D.M.Coen.

ABSTRACT

Acyclovir (ACV), like many antiviral drugs, is a nucleoside analog. In vitro, ACV triphosphate inhibits herpesvirus DNA polymerase by means of binding, incorporation into primer/template, and dead-end complex formation in the presence of the next deoxynucleoside triphosphate. However, it is not known whether this mechanism operates in vivo. To address this and other questions, we analyzed eight mutant polymerases encoded by drug-resistant viruses, each altered in a region conserved among alpha-like DNA polymerases. We measured Km and kcat values for dGTP and ACV triphosphate incorporation and Ki values of ACV triphosphate for dGTP incorporation for each mutant. Certain mutants showed increased Km values for ACV triphosphate incorporation, suggesting a defect in inhibitor binding. Other mutants showed reduced kcat values for ACV triphosphate incorporation, suggesting a defect in incorporation of inhibitor into DNA, while the rest of the mutants exhibited both altered km and kcat values. In most cases, the fold increase in Ki of ACV triphosphate for dGTP incorporation relative to wild-type polymerase was similar to fold resistance conferred by the mutation in vivo; however, one mutation conferred a much greater increase in resistance than in Ki. The effects of mutations on enzyme kinetics could be explained by using a model of an alpha-like DNA polymerase active site bound to primer/template and inhibitor. The results have implications for mechanisms of action and resistance of antiviral nucleoside analogs in vivo, in particular for the importance of incorporation into DNA and for the functional roles of conserved regions of polymerases.

Selected figure(s)

Figure 1.
Fig. 1. Cutaway diagram of an -like DNA polymerase active site containing primer/template and ACV-TP. The -carbon backbone of the protein chain derived from the structure of RB69 DNA polymerase (10) is presented as a gray ribbon diagram. The location of the N terminus of conserved region II is indicated with an arrow. The positions of amino acids altered by HSV drug resistance mutations (e.g., R700G) as well as a conserved serine (S720) relative to the RB69 sequence are indicated with black dots and were aligned according to Wang et al. (10). Primer/template is presented as two ribbons in the upper right quadrant of the figure and ACV-TP is presented as gray dots connected by gray lines.