Eukaryotic replication and chromatin

A single-molecule approach to reveal the dynamics of Eukaryotic DNA replication in chromatin.

Research overview

Replisome in the context of chromatin
The eukaryotic replisome copies DNA that is wrapped about nucleosomes. This state of DNA is also known as chromatin.

The copying, or replication, of DNA is one of the central processes that take place in all living organisms. Our understanding of DNA replication has made gigantic leaps forward since the discovery of the double helical form of DNA by Watson and Crick in 1953. We know many of the structures and functions of the proteins and enzymes involved. Many of these discoveries have been made by studying DNA replication in simple systems, such as viruses or bacteria. These continue to yield valuable insights, but recently, advances in the reconstitution of the yeast replisome have made it possible to gain insights into eukaryotic replication.

DNA replication is carried out at very high accuracy by nanometer-scale, multi-protein complexes known as replisomes. In eukaryotic organisms such as ourselves, the replisome consists of some twenty different proteins. Eukaryotic replication occurs in the context of chromatin: the meters of DNA in eukaryotic organisms are tightly packed into a higher-order structure called chromatin in order to fit in the tiny nucleus. The basic compaction unit of this condensed structure is a DNA-protein complex termed nucleosome which consists of a small piece of DNA wrapped around a core of so-called histone proteins. This compaction adds an extra layer of complexity to the replication process.

Eukaryotic replisome
Schematic of the eukaryotic replisome. The central motor of the replisome is the CMG helicase, which unwinds the parental DNA; the polymerases are then able to add new nucleotides on the leading and lagging strands, respectively, leading to two daughter DNA molecules.

Our research focuses on understanding the molecular processes that underlie eukaryotic DNA replication in the context of chromatin, with the particular aim of gaining spatiotemporal insight into their dynamics by using our single-molecule biophysical expertise in replication and chromatin while integrating it with state-of-the-art molecular biology and biochemistry.

Approach

Despite tremendous advances in understanding chromatin replication achieved by experiments in genetics, cell biology, structural biology, and biochemistry, a detailed mechanistic understanding of how the replisome interacts with nucleosomes, histone chaperones, and chromatin remodelers still remains. The possibility of reconstituting an active yeast replisome in vitro in our lab (originally described by the Diffley laboratory in 2015) has opened up a new perspective, because when integrated with reconstituted forms of chromatin it provides the means to study chromatin replication in a precise, carefully controlled manner.

However, to really understand how the different processes that maintain robust chromatin replication occur in space and time requires probing the stoichiometry and dynamics of the individual proteins involved. Doing so requires a complementary approach to bulk biochemistry that can be found in single-molecule techniques. These high-resolution techniques, which include single-molecule fluorescence and single-molecule force spectroscopy, monitor individual biochemical processes under physiological conditions in real time and have demonstrated their value in revealing the dynamics of large protein complexes.

Read about our recent work

We have recently published our findings about the dynamics of the loading proteins involved in the first step of replisome assembly. The origin recognition complex (ORC) turns out to be a protein that is quite mobile on the DNA, except at the origin. Recruitment of the MCM helicase in an ORC-dependent manner can occur at different locations on the DNA, but immobile ORC-MCM complexes are also preferentially observed at the origin. When loaded onto DNA in the presence of ATP in bulk experiments and then visualized at the single-molecule level, both single and double Mcm2-7 hexamers are found on the DNA, and both exhibit similar low-level mobility. Read about these findings and more here.

Scanning confocal images of two ORC molecules, labelled in green, bound to dsDNA (not fluorescent). The dotted line connects fitted ORC positions over time (blue represents early times; red, late times). In the top panel, the ORC is static throughout the entire observation time bound to the origin of DNA replication. In the bottom panel, another ORC diffuses randomly from its initial position until it locates the origin.

Researchers currently involved in these projects

  • Humberto Sánchez
  • Kaley McCluskey
  • Daniel Ramírez Montero
  • Dorian Mikolajczak
  • Serge Vincent
  • Edo van Veen
  • Theo van Laar

Current collaborators

  • John Diffley Lab (Francis Crick Institute, UK)
  • Francesca Mattiroli Lab (Hubrecht Lab)
  • John van Noort Lab (Leiden University)
  • Alexandra Lusser Lab (University of Innsbruck Medical School, Austria)