Directed evolution of novel polymerase activities: Mutation of a DNA polymerase into an efficient RNA polymerase

Abstract

The creation of novel enzymatic function is of great interest, but remains a challenge because of the large sequence space of proteins. We have developed an activity-based selection method to evolve DNA polymerases with RNA polymerase activity. The Stoffel fragment (SF) of Thermus aquaticus DNA polymerase I is displayed on a filamentous phage by fusing it to a pIII coat protein, and the substrate DNA template͞primer duplexes are attached to other adjacent pIII coat proteins. Phage particles displaying SF polymerases, which are able to extend the attached oligonucleotide primer by incorporating ribonucleoside triphosphates and biotinylated UTP, are immobilized to streptavidin-coated magnetic beads and subsequently recovered. After four rounds of screening an SF library, three SF mutants were isolated and shown to incorporate ribonucleoside triphosphates virtually as efficiently as the wildtype enzyme incorporates dNTP substrates.

Protein evolution with an expanded genetic code

“…Phage display has proven to be a versatile platform for the directed evolution of various protein functions (8)(9)(10)(11)(12)(13). Under the constraints of phage-display evolution, two basic criteria must be met for functional evolution to be successful.…”

A short adaptive path from DNA to RNA polymerases

“…The existence of a second checkpoint for RNA synthesis located in the thumb subdomain had been predicted by us and others (6,10,16,17) on the basis of the phenotype of steric-gate mutants and their premature termination of RNA synthesis, typically at n + 6/7. Indeed, eight of the nine mutations in the starting D4 polymerase (TgoT: L403P, P657T, E658Q, K659H, Y663H, E664Q, D669A, K671N, and T676I) with weak RNA polymerase activity are located in a sequence segment (P657-T676) in the center of the thumb subdomain and in close contact with the nascent strand around n + 6 (Fig.…”

“…The second gate may therefore be a specific adaptation to prevent the deleterious consequences of NTP misincorporation or replication of genomic lesions at high temperature (32). Conversely, despite significant structural variability in the elaboration of the thumb subdomain, extension checkpoints have been observed in different polymerase families, including polA, where similar n + 6 termination of RNA synthesis is observed in steric-gate mutants (16,17). In analogy to the diverse nature of steric-gate residues, the second gate may therefore be elaborated in different ways in the context of different thumb domain structures.…”

“…At the same time, there is compelling structural and phylogenetic evidence for an adaptive path linking DNA to RNA polymerase activity in the evolution of the single-subunit RNA polymerases (ssRNAPs) of mitochondria and T-odd bacteriophages (e.g., T7 RNA polymerase), which are thought to derive from an ancestral polA-family DNA polymerase (19)(20)(21)(22). Thus, we (17) and others (6,10,16) have argued that there must be a determinant of polymerase substrate specificity that has remained unidentified and that precludes synthesis of longer RNAs in the steric-gate mutants.Here, we describe the discovery and characterization of a plausible candidate for such a specificity checkpoint. Using Tgo DNA polymerase, the replicative DNA polymerase from Thermococcus gorgonarius, as our model system, we identify a region in the thumb subdomain, and a single key residue (E664) within it.…”

“…However, although steric-gate mutations generally render DNA polymerases permissive for NTP incorporation, they do not by themselves enable synthesis of RNAs beyond short termination products. Indeed, engineering efforts using rational design (9), in vitro and in vivo screening (14,15), or directed evolution by phage display (16) and compartmentalized self-replication (17) have so far only yielded polymerases that can synthesize RNAs up to 58 nucleotide incorporations (nt) long (14) and more commonly stall at +6-7 nt (14-16, 18) even after prolonged incubation. At the same time, there is compelling structural and phylogenetic evidence for an adaptive path linking DNA to RNA polymerase activity in the evolution of the single-subunit RNA polymerases (ssRNAPs) of mitochondria and T-odd bacteriophages (e.g., T7 RNA polymerase), which are thought to derive from an ancestral polA-family DNA polymerase (19)(20)(21)(22).…”