Dynamics of An Archael DNA Polymerase Revealed By Single Molecule Fret

Abstract

In the archaeon Methanosarcina acetivorans, DNA replication is done by DNA polymerase BI. The processivity of this enzyme is greatly enhanced by a conserved cofactor known as PCNA, which plays a crucial role in orchestrating many replication-related processes. To understand the dynamics of these proteins, we have used single molecule FRET to examine the behavior of PolBI labeled with FRET donor on various DNA structures labeled with FRET acceptor and the effect of PCNA on the dynamics of PolBI. The binding of PolBI to DNA was observed in the low nanomolar concentration range as expected. Interestingly, this polymerase is highly mobile on the DNA structures with two nonadjacent primer strands that are complementary to two different regions in the template strand, 20 nucleotides apart, as evidenced by the frequent transitions between two long-lived FRET states exhibited in single molecule trajectories. To explore the nature of this spontaneous motion, we considered several possible mechanisms including translocations along single- or double-stranded DNA, polymerase binding orientation flipping, and polymerase active site switching. We observed that changes made downstream of the primer/template (P/T) junction have a significant impact on the dynamics of PolBI, indicating the translocation along the single-stranded DNA as the mechanism for the spontaneous motion revealed by FRET. In addition, we have found PCNA not only improves the binding affinity of its cognate polymerase, but suppresses the frequent movement of PolBI from the P/T junction. In summary, many of the dynamics discussed here are reported for the first time and will provide a new perspective for understanding the orchestration of replication-related processes in archaea.