This project aimed at understanding the molecular mechanism of condensin protein using various single-molecule biophysical tools. Condensin is the key protein to organize the chromosome structures in every living cell. It belongs to SMC protein family which are crucial for chromosome organization and segregation. How these SMC proteins organize the chromosome is still a mystery. Recently, in 2018, our group showed DNA loop extrusion mediated by condensin using single-molecule fluorescence assay for the first time. I the current project, I reconstituted DNA loop extrusion using AFM microscopy to understand the mechanism of loop extrusion mediated by condensin. The study is under preparation of a paper submission in Molecular Cell.
Objective 1 (RO1): HS AFM video imaging to watch the structural change of condensin at high spatiotemporal resolution (~ 1 nm, 50 ms).
-. I imaged condensin holocomplex using HS AFM imaging technique with a high spatiotemporal resolution (down to 1 nm and up to 10 frames per second) (Fig. 2 and 3). I observed that the SMC arms of the holocomplex are very flexible (Fig. 3), and there was a transient interaction between hinge region and head domains. Also, we imaged the condensin with/without ATP molecules, and we found that the SMC arms movement occurs with an ATP independent manner. However, ATP binding increased the binding of hinge domain to the head domains (Fig. 4). This suggests that ATP binding induce DNA pulling from hinge domain to the head domains.
Objective 2 (RO2): Real-time visualization of condensin-shaping chromosome using HS AFM.
-. I succeeded in co-imaging condensin and DNA. I reconstituted condensing-mediated DNA loop extrusion on the AFM microscopy (Fig. 1). Also, it shows ATP hydrolysis dependent behaviour. Using both high speed AFM and dry AFM, I could obtain the intermediates of loop extrusion mediated by condensin. I observed that only single condensin localized at the stem of DNA loop. The conformations of condensin at the stem varying, also and the neck size of DNA loop was highly correlated to the width of condensing at the stem. This suggests that the conformational changes of condensin are directly connected to the DNA loop extrusion.
Objective 3 (RO3): Understanding force-generating mechanism of condensin using smFRET-combined magnetic tweezers.
-. Using single-molecule fluorescence assay, I observed single-cohesin’s migration action along the DNA. Labeled cohesin showed that cohesin compacts DNA with an ATP independent manner using clustered cohesin molecules. Also, there was no evidence of ATP dependent behaviour of cohesin, but we observed that cohesin compacts DNA in an ATP independent manner.
