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bioRxiv · 10.64898/2026.10.26.761753

Intercalator effects on DNA loop extrusion by SMC proteins

Analikwu, B. T.·van der Torre, J.·Dekker, C.
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Abstract

Intercalating dyes are widely used to visualize DNA in single-molecule experiments, but their effects on DNA-processing proteins remain incompletely characterized. Here, we study the effects of intercalators on DNA loop extrusion by SMC complexes. Intercalating dyes become fluorescent upon DNA binding, enabling direct visualization of extruded DNA loops. As dye intercalation alters the biophysical properties of DNA by lengthening and unwinding it, we examine how that modifies loop extrusion. We systematically characterize the effects of intercalators, and find that they stiffen DNA, partly neutralize its charge, and can substantially reduce the binding and ATPase activity of SMCs. We assess four dyes and conclude that SYBR Gold and SYTOX Orange are less invasive intercalators than SYTOX Green or YOYO-1. While loop extrusion is qualitatively not affected by intercalation, quantitative values for SMC binding and looping rates differ significantly from those in the absence of intercalators. We present a new dye-free loop-extrusion assay (utilizing local fluorescent spots along DNA), and find that loop extrusion proceeds at 1.75{+/-}0.26 kb/s for S. cerevisiae condensin without dyes, while adding intercalators leads to a two-fold lower rate. Our findings establish that intercalators quantitatively modify loop extrusion parameters, and we offer practical recommendations for minimizing their impact.

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Analikwu, B. T., van der Torre, J., Dekker, C.. 2026-10-26. Intercalator effects on DNA loop extrusion by SMC proteins. https://doi.org/10.64898/2026.10.26.761753

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