2019 Theses Doctoral

Nucleosome Curtains and their application to the study of DNA condensation by condensin

Moevus, Corentin Jean

Single-molecule techniques have the potential to resolve exquisite details inaccessible by common ensemble techniques, such as the dynamic and static hetergeneities of a population. In particular, DNA Curtains, a technique based on total internal reflection fluorescence microscopy, allows for the high-throughput studies of DNA-based processes in real time, at the single-molecule level. The nucleosome, the basic unit of chromatin, is composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Nucleosomes have been extensively studied due to their fundamental inhibitory effect on all eukaryotic DNA-based processes. To study nucleosomes at the single-molecule level using DNA Curtains, a technique based on fluorophore-labeled recombinant nucleosomes was developed. This technique, called Nucleosome Curtains, allows for the study of hundreds of nucleosomes and their interactions with DNA-based processes at the single-molecule resolution, in real-time. Nucleosome Curtains were used to study the effect of nucleosomes on DNA condensation by condensin. Titration of nucleosomes on DNA does not result in slower DNA compaction by condensin, indicating that nucleosomes do not inhibit this process. Direct observation of the interactions between nucleosomes and ongoing DNA compaction by condensin reveals that nucleosomes are readily compacted 80% of the time without any slow-down from condensin. Hence, Nucleosome Curtains is a stable, powerful technique to study nucleosomes at the single-molecule level, and demonstrated that nucleosomes do not affect DNA condensation by condensin.