SV40 Helicase Unwinds DNA against Force

Abstract

Double stranded DNA must be distorted and melted for replication. The large tumor antigen (LTag) of the Simian Virus 40 (SV40) is a simple but robust motor enzyme that opens the DNA duplex, functionally mimicking the eukaryotic replication helicase MCM (MiniChromosome Maintenance). Six monomers of SV40 LTag assemble into a ring around the origin of replication (oriDNA), followed by the formation of a second hexamer. In the presence of ATP and SSB, the two hexamers unwind duplex DNA into single strands. However, the enzyme dynamics and mechanisms largely remain elusive. We use single-molecule magnetic tweezers to investigate the SV40 LTag helicase activity on DNA as a function of force. Magnetic tweezers unwind a torsionally constrained DNA like a helicase, which provides us a convenient way to characterize the DNA response upon unwinding in a broad force range. DNA length changes when unwinding, indicating enzyme activity. From these experiments, we observe that LTag helicase processivity differs between low and high force regimes. We have evaluated the unwinding rate, enzyme pausing, and the effect of SSB and ATP. Our results shed light on the molecular mechanism utilized by the SV40 LTag to open the DNA duplex. Furthermore, our force dependency study indicates how the hexamer-hexamer interactions break during DNA unwinding.