Bacteriophage T4 DNA polymerase mutations that confer sensitivity to the PPi analog phosphonoacetic acid

Abstract

Mutations that conferred sensitivity to the pyrophosphate analog phosphonoacetic acid in bacteriophage T4 DNA polymerase were identified. The mutations were loosely clustered in four regions of the gene. As found for herpes simplex virus DNA polymerase, T4 mutations that altered sensitivity to phosphonoacetic acid also altered sensitivity to nucleotide analogs. Some of the T4 DNA polymerase mutations also altered the ability of the enzyme to translocate from one template position to the next and affected DNA replication fidelity.

Excessive excision of correct nucleotides during DNA synthesis explained by replication hurdles

“…Additionally, the link between the DNAP translocation state and primer-end transfer to the Exo-site is supported by studies of the antimutator strains of bacteriophage T4 DNAP (Reha-Krantz et al, 1993;Reha-Krantz, 1995). A well-studied antimutator, T4 DNAP with mutation A737V, exhibits sensitivity to phosphonoacetic acid, an analog of PPi (Reha-Krantz et al, 1993). This mutant shows excessive excision of correct nucleotides in comparison with wildtype T4 DNAP (Muzyczka et al, 1972) and exhibits difficulty in translocation, particularly on a DNA substrate which requires strand displacement (Gillin & Nossal, 1976).…”

“…Additionally, the link between the DNAP translocation state and primer-end transfer to the Exo-site is supported by studies of the antimutator strains of bacteriophage T4 DNAP (Reha-Krantz et al, 1993;Reha-Krantz, 1995). A well-studied antimutator, T4 DNAP with mutation A737V, exhibits sensitivity to phosphonoacetic acid, an analog of PPi (Reha-Krantz et al, 1993).…”

Genetic characterization of the vaccinia virus DNA polymerase: cytosine arabinoside resistance requires a variable lesion conferring phosphonoacetate resistance in conjunction with an invariant mutation localized to the 3'-5' exonuclease domain

“…Finally, the clustering of the three PAAr lesions strongly suggests that the affected region plays an intimate role in binding the PPi moiety of dNTPs rather than exerting fortuitous effects on protein folding. An alternative mode of action for PAA, in which the drug affects enzyme translocation along the template, has recently been postulated (47). Enzymes with altered PAA sensitivity might pause during translocation; this lag period could alter fidelity by allowing more exonucleolytic proofreading and/or by demanding more stable enzyme-dNTP interactions.…”

Recent Patents of Gene Sequences Relative to DNA Polymerases

“…L412 resides within the highly conserved Motif A in the polymerase active center Fig (4), which has the following amino acid sequence in family B DNA polymerases: DxxSLYPSI, where x is one of several amino acid residues [4,70,[168][169][170]. The phage T4 L412M-DNA polymerase has increased intrinsic processivity compared to the wild type T4 DNA polymerase [171][172][173][174][175], which enables the modified DNA polymerase to efficiently copy difficult DNA sequences including A+T-and G+C-rich DNA sequences and DNA sequences with repeats that cannot be sequenced by other enzymes, for example inverted repeat sequences that produce particularly strong secondary structures (hairpins) [173].…”

“…Because Motif A is highly conserved, the claims for the L412M substitution were extended to similar modifications in other family B DNA polymerases [162,163,174] and to polymerases in general since the aspartate (D) residue in Motif A is conserved in the polymerase active center of all polymerases Fig (4) [169,170]. While protein sequence comparisons and DNA polymerase structures led researchers to Motif A as a likely target to produce variant DNA polymerases that can incorporate non-conventional nucleotides [169,170], in vivo genetic selection strategies led to the discovery of the T4 L412M-DNA polymerase [171,175]. The use of genetic selection methods to identify variant DNA polymerases is discussed in the section on EVOLVING DNA POLYMERASES.…”