Gate dynamics studies of M. smegmatis TOP1A using magnetic tweezers.

Abstract

DNA topoisomerases are enzymes that regulate DNA supercoiling and relieve any structural entanglements that occur during normal cellular processes. We used MT to study the gate dynamics of wild type and mutant Mycobacterium smegmatis (MSM) topoisomerase IA. Two duplex DNA constructs with a 27-nt ssDNA binding region (Gap) were used: one with a 27-nt poly-T tail at the opposite strand and the other without the poly-T tail. A MSM TOP1 binding consensus sequence CCGCTTG was included at the gap region of both constructs. Topoisomerase mediated gate opening and closing were recorded with both wild type and C-terminal deletion mutants at different forces. The force dependent data was analyzed with vbFRET to determine the open and closed states. The states and their lifetimes were then used for kinetic rate analyses. Biased molecular dynamics simulations using metadynamics were also performed on the two MSM structures - with and without ssDNA bound at the topoisomerase catalytic active site. The biasing method was used to explore the open, closed and transition states of the protein mediated DNA gate. We report our findings from both experimental and computational methods which shed light on the working mechanism of MSM TOP1 on a single-molecule scale.