Prof. Nynke Dekker, Oxford University

Prof Nynke Dekker

Title: Unravelling the dynamics of CMG helicase and nucleosomes in chromatin replication using single-molecule approaches

Abstract: During eukaryotic DNA replication, the replisome is assisted by histone chaperones to disassemble parental nucleosomes, and reassemble nucleosomes on the nascent DNA. The replicative helicase, CMG, plays an essential role in this process as it is located at the front of the replisome. Here, we use integrated single-molecule force-fluorescence spectroscopy to monitor the dynamics of Saccharomyces cerevisiae (Sc.) CMG helicase on chromatinized DNA. 
To do so, we use fluorescently labeled Sc. histones to in vitro assemble an individual nucleosome at a nucleosome positioning site on a 14 kb dsDNA substrate. Such chromatinized DNA molecules are then held in an optical trap, and the fluorescent nucleosomes are imaged and subsequently unwrapped through the application of controlled increasing stretching forces to observe the characteristic contour length increment. In this way, we demonstrate that complete nucleosomes with well-controlled positioning are assembled. We then load fluorescently labeled Sc. CMG helicase onto such chromatinized DNA molecules to observe its dynamics through fluorescence imaging. 
Using this approach, we show how the velocity and processivity of CMG helicase are influenced by the encounter with a nucleosome, and conversely how a nucleosome is affected by the encounter with moving CMG helicase. Our findings show that the outcomes depend on the manner in which CMG helicase translocates along the DNA and on the presence of histone chaperones. Future experiments will aim to examine similar processes in the context of the entire replisome to understand the mechanics of chromatin replication.