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Structural maintenance of chromosome (SMC) protein is essential for chromosomal architecture and genome stability across all domains of life. SMC proteins, including cohesin, condensin, and the Smc5/6 complex, are known to organize chromosomes through DNA loop extrusion. However, recent atomic force microscopy (AFM) studies showed ATP-independent DNA-bridging-dependent cohesin/DNA condensate formation, suggesting bridging-induced phase separation (BIPS) for genome organization. Here, we present a comprehensive protocol for AFM image acquisition and data analysis to explore the BIPS induced by cohesin SMC complexes. Since the BIPS mechanism can potentially apply to other multivalent DNA-binding proteins involved in DNA/protein condensate formation, this protocol provides valuable insights into the molecular basis of DNA/protein co-condensation.

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Atomic Force Microscopy Analysis of Bridging-Induced Phase Separation Mediated by SMC Proteins

  • 实验方案

  • 发表日期：

    2026年

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  • Sara Illodo,
  • Hyeong Ku Kim,
  • Je-Kyung Ryu

摘要

Structural maintenance of chromosome (SMC) protein is essential for chromosomal architecture and genome stability across all domains of life. SMC proteins, including cohesin, condensin, and the Smc5/6 complex, are known to organize chromosomes through DNA loop extrusion. However, recent atomic force microscopy (AFM) studies showed ATP-independent DNA-bridging-dependent cohesin/DNA condensate formation, suggesting bridging-induced phase separation (BIPS) for genome organization. Here, we present a comprehensive protocol for AFM image acquisition and data analysis to explore the BIPS induced by cohesin SMC complexes. Since the BIPS mechanism can potentially apply to other multivalent DNA-binding proteins involved in DNA/protein condensate formation, this protocol provides valuable insights into the molecular basis of DNA/protein co-condensation.