Periodic Reporting for period 1 - Cryo-H-Rec (Cryo-EM Imaging of Histone Recycling at the Replication Fork)
Reporting period: 2019-04-01 to 2021-03-31
The main objective of this project is to unravel how replication machinery disassembles nucleosome, how it keeps parental histones close by and how it hands them over to two newly duplicated DNA strands. Epigenetic inheritance is of significant importance for wider society, as it is involved in widespread medical conditions like behavioural disorders, cancers, diabetes and heart problems, as well as in ageing. Understanding molecular mechanism of epigenetic inheritance is required for development of epigenetic therapies that have already shown potential in treatments of certain cancers.
This project has shed some valuable light on the previously unknown mechanism of parental histone recycling. It identified key components of the replication machinery that are responsible for keeping parental histones by its side and offered clues for understanding how they are subsequently handed over behind the replication machinery, towards new DNA strands.
- to express and purify the proteins I am interested in
- to reconstitute full or simplified protein complex pertinent to the biological process I am looking into; in this project that meant reconstituting the core of the replication machinery or sub-complexes that take part in handing over parental histones; in certain experiments I also included DNA in the reconstitution
- after reconstitution, in most cases I performed some kind of purification, so that in the end I get pure complex of interest in my sample
- as all complexes that I have been working on have flexible parts which leads to difficulties in obtaining high-resolution structures with electron microscope, I performed crosslinking of the sample after purification; in this way, the complex is chemically stabilised without affecting its structure
- when the complex is pure and stabilised I would collect a large number of images on the electron microscope
- finally, processing data from the electron microscope would lead to 2D images and 3D representations of the complex that offer valuable information about the arrangement of its subunits, their atomic structure and flexibility; these information are crucial for understanding the mechanism how the complex works
During the project, I established an experimental setup for looking into chromatin replication. Using reconstitution approach described above, I was able to assemble and analyse on the electron microscope several sub-complexes of the replication machinery that include histones and that have not yet been described. These significant findings offer clues for unraveling the mechanism that underlies transfer of parental histones from the original nucleosome in front of the replication machinery towards the newly synthesised DNA behind the replication machinery where they are incorporated into new nucleosomes.
Due to Covid-19 pandemic and some technical difficulties, completion of the project has been delayed. However, I expect to have results for the main objective of the project ready for publication in the first half of next year.
With some delay, I also plan to finalise my work on the mechanism how replication machinery destabilises and disassembles nucleosome when it bumps into it during DNA replication.
Both lines of research are likely to produce high-impact publications during next year.