Single-molecule visualisation of chromatin replication

A Crick PhD position for the 2021 programme in the lab of Hasan Yardimci.

Project background and description

Accurate duplication of the genome is crucial to support proper cell division while maintaining the genetic integrity in all living organisms. Errors in DNA replication can lead to genomic instability, a characteristic feature of most cancer cells. Exploring basic mechanisms of how the replication machinery works and how DNA damage is fixed during replication are pivotal to understanding mechanisms of genome maintenance and have important implications for cancer biology. We use single-molecule fluorescence and force spectroscopy approaches as well as bulk biochemical methods to tackle fundamental mechanistic questions in the eukaryotic DNA replication field. A major advantage of single-molecule methods is that direct observations are made on individual molecules providing unprecedented details about their transient dynamics and heterogeneities in the system.

The eukaryotic DNA replication machinery (replisome) navigates through protein-rich chromatin environment and must overcome various protein obstacles including nucleosomes. Nucleosome consists of ~147 base pairs of DNA wrapped around 8 histone protein cores; central (H3-H4)2 tetramer flanked by two H2A-H2B dimers. During replication, nucleosomes are disrupted ahead of the replication fork, followed by their reassembly on daughter strands from the pool of old parental and newly synthesised histones. Recycling of parental histones at the replication fork is essential to maintain epigenetic inheritance. How parental histones are recycled to daughter strands and how this process is correlated with de novo histone deposition on replicated DNA remain poorly understood. We aim to visualise histone segregation to daughter strands during replication in real time at the single-molecule level to directly address some of the most important questions regarding chromatin replication including

  • Are parental H3-H4 histones transferred behind the replication fork as intact tetramers or are they split into dimers prior to transfer?
  • Are parental histones equally distributed to the leading and lagging strands behind the replication fork?
  • What are the roles of replisome components and histone chaperones in parental histone recycling to either the leading and lagging strands?
  • How parental histone recycling and de novo histone deposition are coordinated? 

To study eukaryotic replication at the single-molecule level we use a number of model systems including Xenopus egg extracts, HeLa cell extracts, and purified yeast and Drosophila proteins. Using novel methodologies that combine single-molecule imaging and conventional ensemble biochemistry, we previously differentiated between different models of unwinding by the replicative DNA helicases [1-4]. Through single-molecule imaging of nucleosomes we recently revealed that the efficiency of parental histone recycling at the replication fork depends on free histone concentration [5].

Candidate background

Consideration will be given to talented and motivated students from various backgrounds including biochemistry, biophysics, physics, and biology.

During the course of graduate studies, the student will develop skills in molecular biology such as cloning, expression and purification of proteins, and single-molecule techniques including total internal fluorescence (TIRF) microscopy and super-resolution fluorescence microscopy.

References

1.       Yardimci, H., Loveland, A.B., Habuchi, S., van Oijen, A.M. and Walter, J.C. (2010)

          Uncoupling of sister replisomes during eukaryotic DNA replication.

          Molecular Cell 40: 834-840. PubMed abstract

2.       Fu, Y.V., Yardimci, H., Long, D.T., Ho, T.V., Guainazzi, A., Bermudez, V.P., . . . Walter, J.C. (2011)

          Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase.

          Cell 146: 931-941. PubMed abstract

3.       Yardimci, H., Loveland, A.B., van Oijen, A.M. and Walter, J.C. (2012)

          Single-molecule analysis of DNA replication in Xenopus egg extracts.

          Methods 57: 179-186. PubMed abstract

4.       Yardimci, H., Wang, X., Loveland, A.B., Tappin, I., Rudner, D.Z., Hurwitz, J., . . . Walter, J.C. (2012)

          Bypass of a protein barrier by a replicative DNA helicase.

          Nature 492: 205-209. PubMed abstract

5.       Gruszka, D.T., Xie, S., Kimura, H. and Yardimci, H. (2020)

          Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication.

          Science Advances 6: eabc0330. PubMed abstract