Structural diversity of supercoiled DNA.

Rossitza N Irobalieva,Wah Chiu,Lynn Zechiedrich,Sarah A Harris,Daniel J Catanese,Anna K Barker,Steven J Ludtke,Michael F Schmid,Thana Sutthibutpong,Jonathan M Fogg,Muyuan Chen

doi:10.1038/ncomms9440

Rossitza N Irobalieva, Wah Chiu + Show 9 more

Open Access

https://doi.org/10.1038/ncomms9440

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Abstract

By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function.

Highlights

By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism
Minicircles containing 336 bp were selected for this study because they are representative of the supercoiled DNA loops found in nature[15,16,17,18]
The two strands of a 336 bp DNA circle wrap around each other 32 times. This number is known as the linking number (Lk)

Summary

Introduction

By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Beyond a sharp supercoiling threshold, we detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Electron microscopy[6,7,8,9,10,11,12,13] (including electron cryo-microscopy6,8,11–13) and atomic force microscopy[10,14] have previously provided significant insight into the structure of negatively supercoiled DNA. Using a combination of approaches we here show that supercoiling causes DNA to adopt large conformational variability. Positive and negative supercoiling are accommodated differently.

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Results

Conclusion