An amino acid residue in the middle of the fingers subdomain is involved in Neq DNA polymerase processivity: enhanced processivity of engineered Neq DNA polymerase and its PCR application.
Neq DNA polymerase is the first archaeal family B DNA polymerase reported to lack uracil recognition function and successfully utilize deaminated bases. We have focused on two amino acid residues (Y515, A523) in the fingers subdomain of Neq DNA polymerase, which were predicted to be located in the middle of the fingers subdomain, based on amino acid sequence alignment of the Neq DNA polymerase with structurally determined archaeal DNA polymerases. Those two residues were replaced by site-directed mutagenesis, and the enzymatic properties of the mutants were analyzed. Here, we show that the A523 residue located in the middle of the fingers subdomain affects the processivity of Neq DNA polymerase. Mutational analysis has allowed us to enhance the protein function as well as understand the function of the residues. One mutant protein, Neq A523R DNA polymerase, exhibited a roughly 3-fold enhanced processivity and extension rate compared to wild type, enabling more efficient PCR. In the presence of uracil, Neq A523R DNA polymerase outperformed Taq DNA polymerase with enhanced specificity and sensitivity. These results suggest that Neq A523R DNA polymerase could be most effectively utilized in real-time PCR using uracil-DNA glycosylase without the risk of carry-over contamination.
Song
Jae-Geun
JG
Department of Genetic Engineering, Sungkyunkwan University, 300 Cheoncheon-Dong, Jangan-Gu, Suwon, Korea.
Kil
Eui-Joon
EJ
Cho
Sung Suk
SS
Kim
In Hye
IH
Kwon
Suk-Tae
ST
D000595:Amino Acid Sequence; D019951:DNA Polymerase beta / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D019641:DNA, Archaeal / Q000378:metabolism*; D015870:Gene Expression; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D046588:Nanoarchaeota / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D016415:Sequence Alignment; D014498:Uracil / Q000378:metabolism
D019641:DNA, Archaeal; D050505:Mutant Proteins; D014498:Uracil; D019951:DNA Polymerase beta