Engineering processive DNA polymerases with maximum benefit at minimum cost

Abstract

DNA polymerases need to be engineered to achieve optimal performance for biotechnological applications, which often require high fidelity replication when using modified nucleotides and when replicating difficult DNA sequences. These tasks are achieved for the bacteriophage T4 DNA polymerase by replacing leucine with methionine in the highly conserved Motif A sequence (L412M). The costs are minimal. Although base substitution errors increase moderately, accuracy is maintained for templates with mono- and dinucleotide repeats while replication efficiency is enhanced. The L412M substitution increases intrinsic processivity and addition of phage T4 clamp and single-stranded DNA binding proteins further enhance the ability of the phage T4 L412M-DNA polymerase to replicate all types of difficult DNA sequences. Increased pyrophosphorolysis is a drawback of increased processivity, but pyrophosphorolysis is curbed by adding an inorganic pyrophosphatase or divalent metal cations, Mn2+ or Ca2+. In the absence of pyrophosphorolysis inhibitors, the T4 L412M-DNA polymerase catalyzed sequence-dependent pyrophosphorolysis under DNA sequencing conditions. The sequence specificity of the pyrophosphorolysis reaction provides insights into how the T4 DNA polymerase switches between nucleotide incorporation, pyrophosphorolysis and proofreading pathways. The L-to-M substitution was also tested in the yeast DNA polymerases delta and alpha. Because the mutant DNA polymerases displayed similar characteristics, we propose that amino acid substitutions in Motif A have the potential to increase processivity and to enhance performance in biotechnological applications. An underlying theme in this chapter is the use of genetic methods to identify mutant DNA polymerases with potential for use in current and future biotechnological applications.

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

“…Thus, pyrophosphorolysis is commonly used to assess the translocation state of RNA polymerases and reverse transcriptases (Marchand & Gotte, ; Hein et al , ). It is known that the DNA sequence itself can influence the equilibrium between pre‐translocation state and post‐translocation states (Tabor & Richardson, ; Reha‐Krantz et al , ), and thus, the pyrophosphorolysis rate constant is sensitive to the particular base‐pair at the primer‐end (Donlin et al , ). If primer‐end binds to the Exo‐site primarily from the pre‐translocation state of the DNAP, then there will be a direct correlation between the rates of the exonuclease and pyrophosphorolysis activities.…”

The RNA triphosphatase domain of L protein of Rinderpest virus exhibits pyrophosphatase and tripolyphosphatase activities

“…Functionally, it is shown that PPi analogues have an inhibitory action on the DNA polymerase d-a ubiquitous eukaryotic polymerase [17]. If the PPi is not removed it would inevitably bind to the postcatalytic polymerase and cause pyrophosphorolysis, thus preventing further replication [18,19]. For the T7 viral RNA polymerase, in vitro studies show that PPi removal is an intrinsic property of the polymerase.…”

Archaeal DNA polymerases in biotechnology

“…The I50L amino acid substitution confers sensitivity to the pyrophosphate-like antiviral drug, phosphonoacetic acid (PAA) as well as a strong mutator phenotype. The L412M substitution in polymerase active center of the T4 DNA pol also causes PAA-sensitivity and reduced fidelity; biochemical studies revealed that the L412M-DNA pol proofread less (Reha-Krantz et al 1993;Reha-Krantz et al 2014). Thus, a working model is that residues in the NPL also function in regulating proofreading (Li et al 2010).…”