Engineered split in Pfu DNA polymerase fingers domain improves incorporation of nucleotide gamma-phosphate derivative.
Using compartmentalized self-replication (CSR), we evolved a version of Pyrococcus furiosus (Pfu) DNA polymerase that tolerates modification of the γ-phosphate of an incoming nucleotide. A Q484R mutation in α-helix P of the fingers domain, coupled with an unintended translational termination-reinitiation (split) near the finger tip, dramatically improve incorporation of a bulky γ-phosphate-O-linker-dabcyl substituent. Whether synthesized by coupled translation from a bicistronic (-1 frameshift) clone, or reconstituted from separately expressed and purified fragments, split Pfu mutant behaves identically to wild-type DNA polymerase with respect to chromatographic behavior, steady-state kinetic parameters (for dCTP), and PCR performance. Although naturally-occurring splits have been identified previously in the finger tip region of T4 gp43 variants, this is the first time a split (in combination with a point mutation) has been shown to broaden substrate utilization. Moreover, this latest example of a split hyperthermophilic archaeal DNA polymerase further illustrates the modular nature of the Family B DNA polymerase structure.
Agilent Technologies Inc., Stratagene Products Division, 11011 N. Torrey Pines Road, La Jolla, CA 92037, USA.
Connie J
CJ
Hansen
Lydia
L
Wu
Jeffrey D
JD
Fox
Bahram
B
Arezi
Holly H
HH
Hogrefe
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D019843:Archaeal Proteins; D009711:Nucleotides; D010710:Phosphates; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase