The functioning of molecular machines: Examining the biochemistry of eukaryotic replication at the single-molecule level

Summary

We each synthesize roughly 5 X 1011 meters of DNA per day – or approximately one light-year’s length of DNA during our lifetimes. Despite this scale, most of us will never develop cancer within our lifetimes. Thus, DNA replication and its associated quality control mechanisms are amazingly robust. How this comes about is, however, from a mechanistic point of view, far from clear.

In eukaryotes such as ourselves, DNA is carried out by a multi-protein complex called the replisome that consists of some twenty different proteins working together as a complex machine to unwind and copy the parental DNA. While the main players that compose the replisome are known, only since 2015 has it become possible to reconstitute their collective activity in vitro1. This presents a very exciting opportunity to address the many open questions that remain about how the different replisome components interact to ensure the rates and processivities that underlie robust DNA replication.

Here, we propose to probe the central motor functioning of the yeast replisome, both on bare DNA and on chromatin. Our goals will be to quantitatively probe the functioning of the key helicase motor protein complex, CMG; to examine how the unwinding of DNA by CMG is impacted by the three types of polymerases that synthesize DNA behind it; to probe the dynamics of these polymerases and any exchange or switching processes they may employ; and to examine how both DNA unwinding by CMG and polymerase dynamics are altered on a chromatin substrate relative to bare DNA. A key feature of our approach that is essential in gaining full insight into the stoichiometry and dynamics of these replisome components during active replication, is that we will employ in our experiments the techniques of single-molecule force spectroscopy and single-molecule fluorescence microscopy. These techniques are particularly well suited to monitoring the presence and motion of proteins in real time at high spatial and temporal resolution, even within protein complexes.

This integrated approach will allow us to understand the functioning of the central motor components of eukaryotic replication and contribute to the general understanding of the robustness of replication in normal cells. It will provide the foundation for subsequent studies linking the impact of replication on epigenetics, transcription, and DNA repair. Understanding replication is of fundamental importance for our comprehension of cell proliferation, and is also a prerequisite to understanding and exploiting the subversion of replication in cancer2,3.

Details

Project number

714.017.002

Main applicant

Prof. dr. N.H. Dekker

Affiliated with

Technische Universiteit Delft, Faculteit Technische Natuurwetenschappen, NanoScience - Kavli Institute of Nanoscience Delft

Duration

01/10/2017 to 01/10/2020