20th-banner-rev.png

Albany 2019: 20th Conversation - Abstracts

category image Albany 2019
Conversation 20
June 11-15 2019
Adenine Press (2019)

Supercoiling Affects Specific Base Accessibility to Alter 3-D Shape of DNA Minicircles

DNA supercoiling affects DNA metabolism yet much about how it does so is unknown (reviewed in Fogg et al. 2012). Using 336 bp DNA minicircles covering a range of positive to negative supercoiling, we unveiled the first three-dimensional structures of supercoiled DNA using cryo-electron tomography (Irobalieva et al. 2015). With supercoiling, DNA can form far more bent and contorted shapes than predicted. We sought to understand how the interplay of DNA sequence and supercoiling drives the formation of these shapes using coarse-grained simulations and biochemical probing. Base pair disruptions indicate regions of high bending, as localized denaturation (molecule dynamic simulations indicate from base flipping; Randall et al. 2009) creates flexible hinges. At the same time, sharp bending at the apices of highly writhed DNA circles leads to broken base pairs. Probing with nuclease Bal-31 revealed exposed bases as a function of supercoiling. Bal31 cleaved all the negatively supercoiled 336 bp minicircles but the rate increased beyond a distinct negative supercoiling threshold. This threshold shifted to more negative supercoiling for 672 bp minicircles with inherently less curvature, demonstrating the relationship between bending and base accessibility. A sharp positive supercoiling threshold was required for Bal-31 cleavage to occur. We mapped Bal-31 cleavage sites and, using coarse-grained simulations, determined the DNA register of our cryo-electron micrograph images. Our data reveal three hotspots of Bal-31 cleavage; two are located ~180º apart along the DNA circumference, and another is seen only in minicircles with low negative supercoiling levels. The relative probability of Bal-31 cleaving at either site varied as a function of supercoiling. Together these data reveal the interplay among sequence, supercoiling, and shape, resulting in conformational changes that should profoundly influence DNA interactions with proteins. Understanding these changes could facilitate the design of supercoiling-dependent DNA nanostructures for gene therapy.

lynn.gif

This work was supported by NIH grant RO1GM115501.

References

    Fogg, J.M., Randall, G.L., Sumners, D.W.L., Pettitt, B.M., Harris, S.A., & Zechiedrich, L. (2012). Bullied no more: when and how DNA shoves proteins around. Quarterly Reviews of Biophysics 45, 257–299

    Irobalieva, R.N.*, Fogg, J.M.*, Catanese, D.J., Sutthibutpong, T., Chen, M., Barker, A.K., Ludtke, S.J., Harris, S.A., Schmid, M.F., Chiu, W. & Zechiedrich, L. (2015) Structural diversity of supercoiled DNA. Nature Communications 12;6:8440 (*co-first authors)

    Randall, G. L., Zechiedrich, L., & Pettitt, B. M. (2009). In the absence of writhe, DNA relieves torsional stress with localized, sequence-dependent structural failure to preserve B-form. Nucleic Acids Research 37, 5568–5577

Jonathan M. Fogg
Lynn Zechiedrich

Department of Molecular Virology and Microbiology
Baylor College of Medicine
Houston, TX 77030

Phone: 713 798 5126
Email: ELZ@BCM.EDU