Periodic Reporting for period 1 - ArcRep (Unravelling the Molecular Specificities of the Archaeal Core Replisome)
Okres sprawozdawczy: 2023-11-01 do 2025-10-31
All cells need to replicate their DNA to divide and survive, which requires an intricate cooperation between multiple proteins that dynamically read the sequence of parental DNA and synthesize new complementary DNA strands. The individual proteins that carry out DNA replication have been extensively studied in recent years, with the aims of fundamentally understanding DNA replication and developing novel molecular biology technologies applicable to the fields of DNA synthesis and next generation sequencing.
The project ArcRep aims to focus on the DNA replication machinery of the archaeon Pyrococcus abyssi, a hyperthermophilic organism isolated from deep sea hydrothermal vents. Its DNA replication proteins resemble those found in eukaryotic cells, while being simpler with a limited set of DNA polymerases and other complementary proteins.
Understanding the interplay between the core replication machinery in Archaea will provide insights into the evolution of DNA replication, and since our model organism is hyperthermophilic, our findings will be applicable to the development of novel molecular biology technologies.
The project ArcRep aims to focus on the DNA replication machinery of the archaeon Pyrococcus abyssi, a hyperthermophilic organism isolated from deep sea hydrothermal vents. Its DNA replication proteins resemble those found in eukaryotic cells, while being simpler with a limited set of DNA polymerases and other complementary proteins.
Understanding the interplay between the core replication machinery in Archaea will provide insights into the evolution of DNA replication, and since our model organism is hyperthermophilic, our findings will be applicable to the development of novel molecular biology technologies.
We have recombinantly expressed and purified all the components of the Pyrococcus abyssi DNA replication machinery, including DNA polymerases (PolD and PolB), the DNA primase (PriSL), the DNA replicative helicase (MCM, GINS and GAN), the processivity factor PCNA and clamp loader RFC, and single-stranded DNA-binding factor RPA.
We have tested multiple combinations of these components to find out which interactions we could observe in vitro, through biophysical methods such as Biolayer Interferometry. We then studied reconstituted protein complexes with an integrative structural biology approach, combining X-ray crystallography, NMR and cryo-EM.
The main achievement of the project has been the reconstitution of the DNA replication components in the lagging strand of the replication fork, comprising the DNA primase (PriSL), DNA polymerase (PolD) and Replication Protein A (RPA). Our work has been published in December 2024 in Nature Communications (https://doi.org/10.1038/s41467-024-55365-w(odnośnik otworzy się w nowym oknie)).
We have also made important progress in the study of the replicative helicase including MCM, GINS and GAN, which will continue after the end of project ArcRep. The scope of the project will be significantly expanded to study the helicase assembly isolated from replicating archaeal cells.
We have tested multiple combinations of these components to find out which interactions we could observe in vitro, through biophysical methods such as Biolayer Interferometry. We then studied reconstituted protein complexes with an integrative structural biology approach, combining X-ray crystallography, NMR and cryo-EM.
The main achievement of the project has been the reconstitution of the DNA replication components in the lagging strand of the replication fork, comprising the DNA primase (PriSL), DNA polymerase (PolD) and Replication Protein A (RPA). Our work has been published in December 2024 in Nature Communications (https://doi.org/10.1038/s41467-024-55365-w(odnośnik otworzy się w nowym oknie)).
We have also made important progress in the study of the replicative helicase including MCM, GINS and GAN, which will continue after the end of project ArcRep. The scope of the project will be significantly expanded to study the helicase assembly isolated from replicating archaeal cells.
Through biolayer interferometry and primer extension assays, we have characterised the interactions between RPA, PriSL and PolD. We have found that the C-terminal domain of the Rpa2 subunit of RPA contacts both PriSL and PolD, and is essential for DNA replication to take place, both in vitro and in vivo in the Thermococcus barophilus model organism. Furthermore, we have described the interaction between RPA and PriSL/PolD in solution through NMR, and we have obtained a crystal structure of the PriS-RPA complex and a cryo-EM structure of the PolD-RPA complex. Taken together, our results show how the dynamic interactions between the lagging strand components of the archaeal replisome perform DNA replication, through a mechanism that is conserved in eukaryotes.
We have also made important progress in the study of the replicative helicase including MCM, GINS and GAN, which will continue after the end of project ArcRep. The scope of the project will be significantly expanded to study the helicase assembly isolated from replicating archaeal cells. To do this, we will recruit an archeal microbiologist for a postdoctoral project in the group.
We have also made important progress in the study of the replicative helicase including MCM, GINS and GAN, which will continue after the end of project ArcRep. The scope of the project will be significantly expanded to study the helicase assembly isolated from replicating archaeal cells. To do this, we will recruit an archeal microbiologist for a postdoctoral project in the group.