Study on biophysical properties of Phi29 DNA polymerase using a novel single-molecule imaging technique DNA curtain.

Abstract

Phi29 DNA polymerase (Phi29 DNAp) is a bacteriophage polymerase that has high fidelity and processivity due to its structural properties. Based on such unique properties, Phi29 DNAp has been used in various biological techniques such as rolling circle amplification and DNA sequencing. However, the detailed mechanism underlying DNA replication by Phi29 DNAp remains elusive. We study the biophysical properties of phi29 DNAp using a high-throughput single-molecule imaging technique DNA curtain. Using DNA curtain, we visualized single replication events in real-time. The speed of Phi29 DNAp is about 130 bp/sec and the processivity is about 23 kbp. Even though the processivity is smaller than that in the previous ensemble measurement, the processivity shows that Phi29 DNAp can synthesize the entire genome of Phi29 bacteriophage in one replication. We also investigate the collision of Phi29 DNAp and protein roadblocks.