Periodic Reporting for period 1 - DNA-Rep-EM (Origin-dependent DNA replication visualised by Cryo-EM)
Reporting period: 2022-09-01 to 2024-08-31
The accurate transfer of genetic information from parental to daughter cells requires that the genome be faithfully copied only once per cell cycle. Failures at any point in this process can lead to cellular abnormalities, genetic disease, and cancer. DNA replication is catalysed by the replisome, a large multi-protein complex that coordinates DNA unwinding and synthesis. A hierarchy of strong and weak functional pair-wise interactions controls and maintains the replisome allowing it to transition through multiple conformational states. These transitions are intimately linked to a small number of essentially irreversible chemical steps (ATP-hydrolysis, RNA-primer and DNA synthesis), which determine reaction directionality. In vitro reconstitution of DNA replication with purified yeast proteins has helped uncover important mechanisms of DNA replication. To date, structural studies have focused on imaging artificially isolated replication complexes using simplified DNA substrates to understand DNA unwinding and replisome architecture. To truly understand the mechanisms that control DNA replication, future structural studies must not only visualise isolated complexes, but also reconstituted reactions. This project aims to visualise origin-dependent eukaryotic DNA replication using in vitro reconstituted cellular reactions in their entirety, at near atomic resolution. These data will gain a deeper understanding of the molecular mechanisms that permit the eukaryotic replisome to function during genome duplication.
This project has shed some valuable light on the previously unknown mechanism of origin DNA unwinding and spatial orientation of key replisome components.
This project has shed some valuable light on the previously unknown mechanism of origin DNA unwinding and spatial orientation of key replisome components.
This project aimed to use cryo-electron microscopy and state-of-the-art biochemistry to study the molecular mechanism of origin activation and replisome progression using purified yeast proteins. I leveraged a comprehensive suite of biochemical, structural biology, and computational resources available at the Costa lab and the Francis Crick Institute. My project consisted of three overarching aims:
1. Reconstitution of origin activation on short DNA substrates for cryo-EM – I developed assays to robustly study eukaryotic origin activation using cryo-EM. We have submitted a manuscript showing the structural role of Mcm10 during origin activation and unwinding.
2. Determine the mechanism of lagging-strand priming at an origin of replication – Work on this aim faced delays and had to be significantly altered due to early termination of the fellowship and results published from another lab detailing the structure of Pol alpha primase engaged with the replicative helicase.
3. Understand how DNA synthesis is coordinated on the leading and lagging strands capturing fork progression at high-resolution – We have established an approach to study a minimal leading strand replisome and have preliminary cryo-EM structures which will ultimately help us understand how DNA unwinding and polymerisation are coupled.
1. Reconstitution of origin activation on short DNA substrates for cryo-EM – I developed assays to robustly study eukaryotic origin activation using cryo-EM. We have submitted a manuscript showing the structural role of Mcm10 during origin activation and unwinding.
2. Determine the mechanism of lagging-strand priming at an origin of replication – Work on this aim faced delays and had to be significantly altered due to early termination of the fellowship and results published from another lab detailing the structure of Pol alpha primase engaged with the replicative helicase.
3. Understand how DNA synthesis is coordinated on the leading and lagging strands capturing fork progression at high-resolution – We have established an approach to study a minimal leading strand replisome and have preliminary cryo-EM structures which will ultimately help us understand how DNA unwinding and polymerisation are coupled.
This work is state of the art cryo-EM and biochemistry; the reagents and assays developed herein will allow researchers to truly understand the mechanisms that control DNA replication, future structural studies must not only visualise isolated complexes, but also reconstituted reactions. While some outcomes are delayed due to international relocation and early termination of the fellowship some, ongoing work and data processing are likely to produce high-impact publications during next year including a co-corresponding methods paper. Since errors in the mechanisms that control DNA replication can cause genomic instability and lead to the development of genetic diseases and cancer, our work may explain the molecular basis of some human diseases.