Engineered split in Pfu DNA polymerase fingers domain improves incorporation of nucleotide  -phosphate derivative

Abstract

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.

Modified Nucleoside Triphosphates for In-vitro Selection Techniques

“… DNAP, DNA polymerase; RNAP, RNA polymerase; NTP, nucleoside triphosphate; CeNA, cyclohexenyl nucleic acid; LNA, locked nucleic acid; ANA, arabino nucleic acid; FANA, 2′-F-arabino nucleic acid; HNA, 1,5-anhydrohexitol nucleic acid; d5NI, 5-nitroindole; d5NIC, 5-nitroindole-3-carboxamide; ICS, isocarbostyril; 7AI, 7-azaindole. Refrences: 1 (Padilla and Sousa, 1999 ); 2 (Raines and Gottlieb, 1998 ); 3 (Padilla and Sousa, 2002 ); 4 (Burmeister et al, 2006 ); 5 (Chelliserrykattil and Ellington, 2004 ); 6 (Patel and Loeb, 2000 ); 7 (Patel et al, 2001 ); 8 (Suzuki et al, 1996 ); 9 (Ogawa et al, 2001 ); 10 (Ong et al, 2006 ); 11 (Xia et al, 2002 ); 12 (Fa et al, 2004 ); 13 (Schultz et al, 2015 ); 14 (Shinkai et al, 2001 ); 15 (Pinheiro et al, 2012 ); 16 (Ghadessy et al, 2004 ); 17 (Laos et al, 2013 ); 18 (Loakes et al, 2009 ); 19 (Leconte et al, 2005 ); 20 (Ramsay et al, 2010 ); 21 (Hansen et al, 2011 ). …”

“…In another study, the M1 variant of Taq DNA polymerase allowed the full substitution of dNTPs with phosphorothioates (Ghadessy et al, 2004 ). Regarding less frequent modifications, T7 RNA polymerase was used to select an ATP-binding aptamer containing 5′-(α-P-borano)-GTP and 5′-(α-P-borano)-UTP (Lato et al, 2002 ) and a mutated Pfu DNA polymerase was able to incorporate nucleotides bearing a bulky γ-phosphate-O-linker-dabcyl substituent (Hansen et al, 2011 ).…”

Directed polymerase evolution

“…Polymerases are usually specific for triphosphate monomers, however, some applications would be facilitated by the synthesis of DNA or RNA from monomers with modified triphosphate moieties. Hansen et al evolved the family B DNAP from Pyrococcus furiosus ( Pfu ) for improved incorporation of nucleotides with modified γ‐phosphates upon library construction by epPCR and CSR selection [73]. A variant bearing the Q484R mutation and an internal split (resulting from the addition of a premature stop codon and the re‐initiation of transcription downstream, and thus the production of two fragments that associate to form the active DNAP) was identified that gained the ability to incorporate γ‐phosphate‐ O ‐linker‐dabcyl derivatives.…”

“…Baar et al also evolved a DNAP for enhanced resistance to inhibitors by constructing a library via family shuffling of Pol I genes and CSR selection in the presence of inhibitors [72]. A chimera of T. aquaticus, Thermus oshimai, Ther- [73]. A variant bearing the Q484R mutation and an internal split (resulting from the addition of a premature stop codon and the re-initiation of transcription downstream, and thus the production of two fragments that associate to form the active DNAP) was identified that gained the ability to incorporate c-phosphate-O-linker-dabcyl derivatives.…”

Exploring the Chemistry of Genetic Information Storage and Propagation through Polymerase Engineering

“…55 CSR also proved useful in evolving polymerases for the expansion of the genetic alphabet by improved incorporation of the unnatural base dZTP opposite its cognate dP template ( 3 ). 56 In a similar approach CSR was used to evolve RT activity using chimeric RNA–DNA primers requiring reverse transcription through the RNA portion for self-replication.…”