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Engineering processive DNA polymerases with maximum benefit at minimum cost.

Reha-Krantz LJ, Woodgate S, Goodman MF - Front Microbiol (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Alberta Edmonton, AB, Canada.

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, Mn(2+) or Ca(2+). 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.

No MeSH data available.


Related in: MedlinePlus

Inhibiting pyrophosphorolysis with manganese. Increased manganese is needed to inhibit the pyrophosphorolysis reaction catalyzed by the exonuclease-deficient L412M-DNA polymerase when the concentration of PPi is increased.
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Figure 4: Inhibiting pyrophosphorolysis with manganese. Increased manganese is needed to inhibit the pyrophosphorolysis reaction catalyzed by the exonuclease-deficient L412M-DNA polymerase when the concentration of PPi is increased.

Mentions: Na citrate protects the nucleotide incorporation reaction by functioning as a weak chelator, which reversibly binds divalent metal cations; however, PPi also forms complexes with metal cations. This point is demonstrated by the observation that higher amounts of Mn2+ ions are needed to inhibit pyrophosphorolysis by the T4 L412M-DNA polymerase as the concentration of PPi is increased (Damaraju and Reha-Krantz). For example, in the presence of 1 mM PPi, ∼ 1 mM Mn2+ produces 50% inhibition of pyrophosphorolysis; for 2 mM PPi, ∼2 mM Mn2+ is needed for 50% inhibition; and for 4 mM PPi, >3 mM Mn2+ is required to achieve the same level of inhibition (Figure 4). The stoichiometry of the reaction is consistent with formation of Mn-PPi chelate complexes or in the case of Ca2+, Ca-PPi chelate complexes. Thus, there is a complex equilibria involving one or more metal ions (Me): DNA pol-Me, citrate-Me, PPi- Me, free Me2+, free DNA pol, free PPi, free Na citrate, and active and inactive DNA pol- Me-PPi complexes. While Mn-PPi binding may inactivate pyrophosphorolysis directly, this is not the most likely explanation for inhibition in reactions with Na citrate and Mn2+ above 3 mM because nucleotide incorporation is not inhibited; Mn-PPi binding would be expected to inhibit both reactions. Another explanation is that Mn2+ sequesters PPi and reduces the high concentration of PPi needed for the pyrophosphorolysis reaction, but there is sufficient free Mn2+ (∼0.5 mM) to support, but not to inhibit, the nucleotide incorporation reaction. The ability of Na citrate to maintain low concentrations of Mn2+ has been proposed by others (Beckman et al., 1985; Tabor and Richardson, 1989, 1990), but in this case Mn2+ was thought to improve the evenness of band intensities (DNA sequencing products) by reducing discrimination against chain-terminating nucleotides. Ca2+ can also chelate PPi and effectively prevent pyrophosphorolysis (results not shown).


Engineering processive DNA polymerases with maximum benefit at minimum cost.

Reha-Krantz LJ, Woodgate S, Goodman MF - Front Microbiol (2014)

Inhibiting pyrophosphorolysis with manganese. Increased manganese is needed to inhibit the pyrophosphorolysis reaction catalyzed by the exonuclease-deficient L412M-DNA polymerase when the concentration of PPi is increased.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4120765&req=5

Figure 4: Inhibiting pyrophosphorolysis with manganese. Increased manganese is needed to inhibit the pyrophosphorolysis reaction catalyzed by the exonuclease-deficient L412M-DNA polymerase when the concentration of PPi is increased.
Mentions: Na citrate protects the nucleotide incorporation reaction by functioning as a weak chelator, which reversibly binds divalent metal cations; however, PPi also forms complexes with metal cations. This point is demonstrated by the observation that higher amounts of Mn2+ ions are needed to inhibit pyrophosphorolysis by the T4 L412M-DNA polymerase as the concentration of PPi is increased (Damaraju and Reha-Krantz). For example, in the presence of 1 mM PPi, ∼ 1 mM Mn2+ produces 50% inhibition of pyrophosphorolysis; for 2 mM PPi, ∼2 mM Mn2+ is needed for 50% inhibition; and for 4 mM PPi, >3 mM Mn2+ is required to achieve the same level of inhibition (Figure 4). The stoichiometry of the reaction is consistent with formation of Mn-PPi chelate complexes or in the case of Ca2+, Ca-PPi chelate complexes. Thus, there is a complex equilibria involving one or more metal ions (Me): DNA pol-Me, citrate-Me, PPi- Me, free Me2+, free DNA pol, free PPi, free Na citrate, and active and inactive DNA pol- Me-PPi complexes. While Mn-PPi binding may inactivate pyrophosphorolysis directly, this is not the most likely explanation for inhibition in reactions with Na citrate and Mn2+ above 3 mM because nucleotide incorporation is not inhibited; Mn-PPi binding would be expected to inhibit both reactions. Another explanation is that Mn2+ sequesters PPi and reduces the high concentration of PPi needed for the pyrophosphorolysis reaction, but there is sufficient free Mn2+ (∼0.5 mM) to support, but not to inhibit, the nucleotide incorporation reaction. The ability of Na citrate to maintain low concentrations of Mn2+ has been proposed by others (Beckman et al., 1985; Tabor and Richardson, 1989, 1990), but in this case Mn2+ was thought to improve the evenness of band intensities (DNA sequencing products) by reducing discrimination against chain-terminating nucleotides. Ca2+ can also chelate PPi and effectively prevent pyrophosphorolysis (results not shown).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Alberta Edmonton, AB, Canada.

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, Mn(2+) or Ca(2+). 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.

No MeSH data available.


Related in: MedlinePlus