Variants of sequence family B Thermococcus kodakaraensis DNA polymerase with increased mismatch extension selectivity

Abstract

Fidelity and selectivity of DNA polymerases are critical determinants for the biology of life, as well as important tools for biotechnological applications. DNA polymerases catalyze the formation of DNA strands by adding deoxynucleotides to a primer, which is complementarily bound to a template. To ensure the integrity of the genome, DNA polymerases select the correct nucleotide and further extend the nascent DNA strand. Thus, DNA polymerase fidelity is pivotal for ensuring that cells can replicate their genome with minimal error. DNA polymerases are, however, further optimized for more specific biotechnological or diagnostic applications. Here we report on the semi-rational design of mutant libraries derived by saturation mutagenesis at single sites of a 3'-5'-exonuclease deficient variant of Thermococcus kodakaraensis DNA polymerase (KOD pol) and the discovery for variants with enhanced mismatch extension selectivity by screening. Sites of potential interest for saturation mutagenesis were selected by their proximity to primer or template strands. The resulting libraries were screened via quantitative real-time PCR. We identified three variants with single amino acid exchanges-R501C, R606Q, and R606W-which exhibited increased mismatch extension selectivity. These variants were further characterized towards their potential in mismatch discrimination. Additionally, the identified enzymes were also able to differentiate between cytosine and 5-methylcytosine. Our results demonstrate the potential in characterizing and developing DNA polymerases for specific PCR based applications in DNA biotechnology and diagnostics.

Modified Taq DNA Polymerase for Allele-Specific Ultra-Sensitive Detection of Genetic Variants

“…There have been previous efforts to improve the function of DNA polymerases by changing amino acids that affect the interaction between the polymerase and the primer–template complex. 19 , 24 , 25 , 26 For example, substitution of the positively charged amino acids (R536, R587, or R660) of the Taq DNA polymerase Klenow fragment, which are directly in contact with the phosphate backbone of the primer in the closed conformation, has been shown to increase primer selectivity. 19 However, when the single point mutations at these locations were combined to improve the selectivity, a drop in polymerase activity was observed.…”

Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library

“…Screening was performed with heat-denatured bacterial lysates as successful as before to identify active and inactive mutants using real time PCR 28 . For this purpose, a 92mer template DNA of the NANOG promoter region 29,30 along with corresponding primers and SYBR ® Green I were employed in screening reaction and the data was recorded. The PCR active and inactive mutants were determined by measuring the respective Cq value, the cycle number at which the fluorescence signal crosses the background signal with an exponential increase of the signal.…”

Direct Enzyme Engineering of B Family DNA Polymerases for Biotechnological Approaches

“…Exovariant + Gly245Asp 5-methyl-cytosine sensitive [72] Exovariant + Arg501Cys, Arg606Gln/Trp Mismatches/5-methyl-cytosine sensitive [73] K.RT521K: Val93Glu, Asp141Ala, Glu143Ala, Ala485Leu, Ile521Leu, Glu664Lys Reverse synthesis of DNA on tPhoNA strand [63] RTX: Phe38Leu, Arg97Met, Lys118Ile, Met137Leu, Arg381His, Tyr384His, Val389Ile, Lys466Arg, Tyr493Leu, Thr514Ile, Ile521Leu, Phe587Leu, Glu664Lys, Gly711Val, Asn735Lys, Trp768Arg.…”

“…In [73], other mutant forms of KOD-exo − , namely, Arg501Cys, Arg606Gln, and Arg606Trp, are described that show increased sensitivity to the presence of mismatches and can discriminate between C and 5 mC. The substitution of Arg606 led to an increase in the number of selective contacts with a primer, whereas the substitution of Arg501 resulted in an increase in the number of selective contacts with the template owing to the removal of positive charges, which probably stabilize the interactions of the enzyme with the primer-template duplex, regardless of whether it contains a mismatch.…”