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. 2004 Dec 15;32(22):e176.
doi: 10.1093/nar/gnh174.

Low-fidelity Pyrococcus Furiosus DNA Polymerase Mutants Useful in Error-Prone PCR

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Low-fidelity Pyrococcus Furiosus DNA Polymerase Mutants Useful in Error-Prone PCR

Benjamin D Biles et al. Nucleic Acids Res. .
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Abstract

Random mutagenesis constitutes an important approach for identifying critical regions of proteins, studying structure-function relations and developing novel proteins with desired properties. Perhaps, the most popular method is the error-prone PCR, in which mistakes are introduced into a gene, and hence a protein, during DNA polymerase-catalysed amplification cycles. Unfortunately, the relatively high fidelities of the thermostable DNA polymerases commonly used for PCR result in too few mistakes in the amplified DNA for efficient mutagenesis. In this paper, we describe mutants of the family B DNA polymerase from Pyrococcus furiosus (Pfu-Pol), with superb performance in error-prone PCR. The key amino acid changes occur in a short loop linking two long alpha-helices that comprise the 'fingers' sub-domain of the protein. This region is responsible for binding the incoming dNTPs and ensuring that only correct bases are inserted opposite the complementary base in the template strand. Mutations in the short loop, when combined with an additional mutation that abolishes the 3'-5' proof-reading exonuclease activity, convert the extremely accurate wild-type polymerase into a variant with low fidelity. The mutant Pfu-Pols can be applied in error-prone PCR, under exactly the same conditions used for standard, high-fidelity PCR with the wild-type enzyme. Large quantities of amplified product, with a high frequency of nearly indiscriminate mutations, are produced. It is anticipated that the Pfu-Pol variants will be extremely useful for the randomization of gene, and hence protein, sequences.

Figures

Figure 1
Figure 1
(A) Structure of the fingers sub-domain of polymerase 9°N-7. The sub-domain is made up of two long α-helices (the N and O-helices), separated by a short loop (amino acids in the loop shown in green). Folding of the sub-domain brings a number of highly conserved amino acids, shown in red, into close proximity on the same side of the motif. The red and green amino acids in this panel correspond to the identically coloured amino acid for the second entry (Therm. 9°N-7 sequence) in (B). (B) Amino acid alignment of the fingers sub-domain of a number of archaeal family-B DNA polymerases (accession numbers given in brackets). The highly conserved amino acids are shown in red, as in (A). Amino acids that form the loop between the two helices are shown in green. For clarity, only sequences that can be lined up without the introduction of gaps (many polymerases have a few extra amino acids in the loop region, necessitating gaps) have been included. Abbreviations: Pyr, Pyrococcus; Therm, Thermococcus; Methsar, Methanosarcina; Methcoc, Methanococcoides; Ferro, Ferroplasma; Meththerm thermauto, Methanothermobacter thermautotrophicus; Sulfur, Sulfurisphaera; and Desulfur, Desulforococcus.
Figure 2
Figure 2
(A) PCR amplification of a 1.9 kb lacIOZα sequence by Pfu-Pol. The lanes containing four bands, separating the different Pfu-Pol mutants, are length markers of 3, 2, 1.5 and 1 kb. (B) Pfu-Pol catalysed extension of the primer strand of a primer-template in the presence of a single dNTP, either dATP (left of dotted line) or dGTP (right of dotted line). The primer-template possesses the structure: 5′-[32P]pGGCGCCCGCGG/3′-CCGCGGGCGCCTCGAAG. The primer runs at the position indicated ‘N’; addition of a first and second dNTP gives the extended products running at ‘N+1’ and ‘N+2’, respectively. The time points are 0, 7, 14, 35, 60 and 300 s.
Figure 3
Figure 3
Alterations in DNA found after error-prone PCR using Pfu-Pol(exo)D473G. The nomenclature A-G indicates a change in the sequenced strand from adenine to guanine. The actual numbers observed for each alteration are given in brackets; the arrow indicates the frequency expected (8.3%) for completely random changes (excluding insertions and deletions). As explained in the text, certain substitutions, e.g. T-G and A-C, are equivalent and their frequencies need to be pooled for analysis of bias (Table 2).
Figure 4
Figure 4
Close up of the loop region of polymerase 9°N-7, comprising the three loop amino acids TVD (green) and the four flanking amino acids from both the N α-helix (cyan) and the O α-helix (magenta) (see Figure 1B, second entry for the identification of these amino acids). The loop aspartic acid (side chain and backbone carbonyl) makes a network of hydrogen bonds (black dashed lines) to the backbone NH group of three amino acids (LEK) in the O-helix. The middle valine of the loop does not make any interactions with the rest of the protein and the side chain points towards the solvent. The loop threonine makes a single hydrogen bond to a glutamic acid in the O-helix.

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