In vitro evolution of phi29 DNA polymerases through compartmentalized gene expression and rolling-circle replication

Phi29 DNA polymerase is widely used for DNA amplification through rolling-circle replication or multiple displacement amplification. Here, we performed completely in vitro artificial evolution of phi29 DNA polymerase by combining the in vitro compartmentalization and the gene expression-coupled rolling-circle replication of a circular DNA encoding the polymerase. We conducted the experiments in six different conditions composed of three different levels of inhibitor concentrations with two different DNA labeling methods. One of the experiments was performed in our previous study and the other five experiments were newly conducted in this study. Under all conditions, we found several mutations that enhance the rolling-circle amplification by the polymerase when it was expressed in the reconstituted gene expression system. Especially, a combinatorial mutant polymerase (K555T/D570N) exhibits significantly higher rolling-circle activity than the wild type. These highly active mutant polymerases would be useful for various applications.

The Loop of the TPR1 Subdomain of Phi29 DNA Polymerase Plays a Pivotal Role in Primer-Terminus Stabilization at the Polymerization Active Site

Bacteriophage Phi29 DNA polymerase belongs to the protein-primed subgroup of family B DNA polymerases that use a terminal protein (TP) as a primer to initiate genome replication. The resolution of the crystallographic structure showed that it consists of an N-terminal domain with the exonuclease activity and a C-terminal polymerization domain. It also has two subdomains specific of the protein-primed DNA polymerases; the TP Regions 1 (TPR1) that interacts with TP and DNA, and 2 (TPR2), that couples both processivity and strand displacement to the enzyme. The superimposition of the structures of the apo polymerase and the polymerase in the polymerase/TP heterodimer shows that the structural changes are restricted almost to the TPR1 loop (residues 304–314). In order to study the role of this loop in binding the DNA and the TP, we changed the residues Arg306, Arg308, Phe309, Tyr310, and Lys311 into alanine, and also made the deletion mutant Δ6 lacking residues Arg306–Lys311. The results show a defective TP binding capacity in mutants R306A, F309A, Y310A, and Δ6. The additional impaired primer-terminus stabilization at the polymerization active site in mutants Y310A and Δ6 allows us to propose a role for the Phi29 DNA polymerase TPR1 loop in the proper positioning of the DNA and TP-priming 3’-OH termini at the preinsertion site of the polymerase to enable efficient initiation and further elongation steps during Phi29 TP-DNA replication.

Robust and highly specific fluorescence sensing of Salmonella typhimurium based on dual-functional phi29 DNA polymerase-mediated isothermal circular strand displacement polymerization

A simple and robust fluorescence sensing strategy has been developed for the detection of pathogenic bacteria by the combination of the dual functionality of phi29 DNA polymerase with isothermal circular strand displacement polymerization (ICSDP).