Direct single-molecule observations of DNA unwinding by SV40 large tumor antigen under a negative DNA supercoil state

Abstract

Superhelices, which are induced by the twisting and coiling of double-helical DNA in chromosomes, are thought to affect transcription, replication, and other DNA metabolic processes. In this study, we report the effects of negative supercoiling on the unwinding activity of simian virus 40 large tumor antigen (SV40 TAg) at a single-molecular level. The supercoiling density of linear DNA templates was controlled using magnetic tweezers and monitored using a fluorescent microscope in a flow cell. SV40 TAg-mediated DNA unwinding under relaxed and negative supercoil states was analyzed by the direct observation of both single- and double-stranded regions of single DNA molecules. Increased negative superhelicity stimulated SV40 TAg-mediated DNA unwinding more strongly than a relaxed state; furthermore, negative superhelicity was associated with an increased probability of SV40 TAg-mediated DNA unwinding. These results suggest that negative superhelicity helps to regulate the initiation of DNA replication.

Keywords:

• single-molecule observation
• negative supercoil
• DNA unwinding
• SV40 large tumor antigen
• initiation of DNA replication

Acknowledgment

We thank Mr Akio Fujie and Drs Kaoru Sugasawa and Fumio Yatagai for the purification of SV40 TAg.