Periodic Reporting for period 1 - PRIMASE_NMR (Visualizing Primase Initiating DNA Replication using NMR Spectroscopy)
Reporting period: 2016-03-01 to 2018-02-28
The genetic information encoded in DNA in a fundamentally important element of any living organism. A key aspect of DNA is its capability to be replicated, allowing for the transfer of the genetic information upon generations of cells or organisms. It is therefore of uttermost importance to understand the detailed mechanism of DNA replication. DNA replication happens in three steps. First, the helicases unwind the DNA to make it accessible to other biological actors. Then, primases interact with the DNA strands to synthesize short primers that will finally be extended by polymerases to copy the full genetic information.
DNA is made of building blocks, the nucleotides, and DNA transcription corresponds to assemble nucleotides in the correct order to ensure the exact duplication of the initial DNA strand. Polymerases are able to add an extra nucleotide to an existing DNA strand but primases have the unique ability to assemble nucleotides from scratch and in particular to assemble together the two first nucleotides. This step of initiation is absolutely key in the DNA replication process as any further duplication procedures rely on this initial step.
However, up to now, the detailed mechanism by which primases act remains quite elusive. To understand how the initiation of replication appends, it is necessary to understand how those nucleotides interact with the primase at the atomic level. To do so, we used Nuclear Magnetic Resonance (NMR) spectroscopy, the technique of choice to study biomolecular transient interactions at the atomic resolution.
Using NMR spectroscopy our objectives are to understand: (i) how the primase and the DNA interact with nucleotides, (ii) how this interactions influence the properties of the primase, the DNA and the nucleotides, (iii) how it is possible to create the first bond between two nucleotides and (iv) how this dinucleotides can be extended into a short primer of defined length.
Answering those questions will have a strong impact on our fundamental understanding of DNA replication. Due to the essential role of DNA replication, the better understanding of this process will help in the development of novel biological or biomedical techniques related to this mechanism.
DNA is made of building blocks, the nucleotides, and DNA transcription corresponds to assemble nucleotides in the correct order to ensure the exact duplication of the initial DNA strand. Polymerases are able to add an extra nucleotide to an existing DNA strand but primases have the unique ability to assemble nucleotides from scratch and in particular to assemble together the two first nucleotides. This step of initiation is absolutely key in the DNA replication process as any further duplication procedures rely on this initial step.
However, up to now, the detailed mechanism by which primases act remains quite elusive. To understand how the initiation of replication appends, it is necessary to understand how those nucleotides interact with the primase at the atomic level. To do so, we used Nuclear Magnetic Resonance (NMR) spectroscopy, the technique of choice to study biomolecular transient interactions at the atomic resolution.
Using NMR spectroscopy our objectives are to understand: (i) how the primase and the DNA interact with nucleotides, (ii) how this interactions influence the properties of the primase, the DNA and the nucleotides, (iii) how it is possible to create the first bond between two nucleotides and (iv) how this dinucleotides can be extended into a short primer of defined length.
Answering those questions will have a strong impact on our fundamental understanding of DNA replication. Due to the essential role of DNA replication, the better understanding of this process will help in the development of novel biological or biomedical techniques related to this mechanism.
The studied Archaeal primase (pRN1), contains two domains, a helix bundle domain that binds DNA and a catalytic domain that encompasses the catalytic site. The helix bundle domain as been studied in the lab for many years thus our focus has been on the catalytic domain and on the full-length primase.
In order to be investigated by NMR spectroscopy, a protein has to be produced isotopically labeled in large quantities. In that perspective, we have established procedures to produce NMR quantities of those systems with a variety of labeling scheme (uniformly 15N, 13C and/or 2H and specific unlabelling or particular amino-acids).
Due to the large size of this system, the primase needs to be studied at high temperature to ensure sufficient spectral qualities (40-50C). This is easily feasible for the protein as it comes from a thermophilic organism, but is a major problem for nucleotides triphosphates (NTPs) as they quickly hydrolyze at those temperatures. Thus, a strategy based on an in situ recycling system was developed and optimized to allow working routinely at 40C and thus opening novel avenues to study this system.
To allow for site-specific information by NMR the spin system of the protein needs to be assigned, which has been achieved here for the catalytic domain, in absence of NTPs at 40 and 50C and in presence of NTPs at 40C.
As the crystal structure of this system is available additional information were perform to test and reveal that this structure is still a good model for the catalytic domain under those conditions.
Then, the catalytic domain interactions with several different NTPs were investigated. In particular, regions of interactions have been identified. Additional structural and dynamic changes have been investigated using state-of-the-art NMR approaches that underline a complex mechanism of interaction between the NTPs and the primase.
This information has also been replaced within the context of the full-length protein and used to better understand the interplay between the two domains.
Finally solid-state NMR studies of the full-length protein have been achieved to provide a complementary vision on the same processes.
In terms of dissemination of the results several publications are planned, at least:
- one publication relative to the DNA recognition by the helix bundle domain and how this effect happens within the full length
- one publication on a novel alignment media that has been developed to obtained structural and dynamic information about the primase and the catalytic domain at high temperature
- one publication about how the catalytic domain interact with nucleotides and how it influences our understanding of the catalytic cycle of this system
In order to be investigated by NMR spectroscopy, a protein has to be produced isotopically labeled in large quantities. In that perspective, we have established procedures to produce NMR quantities of those systems with a variety of labeling scheme (uniformly 15N, 13C and/or 2H and specific unlabelling or particular amino-acids).
Due to the large size of this system, the primase needs to be studied at high temperature to ensure sufficient spectral qualities (40-50C). This is easily feasible for the protein as it comes from a thermophilic organism, but is a major problem for nucleotides triphosphates (NTPs) as they quickly hydrolyze at those temperatures. Thus, a strategy based on an in situ recycling system was developed and optimized to allow working routinely at 40C and thus opening novel avenues to study this system.
To allow for site-specific information by NMR the spin system of the protein needs to be assigned, which has been achieved here for the catalytic domain, in absence of NTPs at 40 and 50C and in presence of NTPs at 40C.
As the crystal structure of this system is available additional information were perform to test and reveal that this structure is still a good model for the catalytic domain under those conditions.
Then, the catalytic domain interactions with several different NTPs were investigated. In particular, regions of interactions have been identified. Additional structural and dynamic changes have been investigated using state-of-the-art NMR approaches that underline a complex mechanism of interaction between the NTPs and the primase.
This information has also been replaced within the context of the full-length protein and used to better understand the interplay between the two domains.
Finally solid-state NMR studies of the full-length protein have been achieved to provide a complementary vision on the same processes.
In terms of dissemination of the results several publications are planned, at least:
- one publication relative to the DNA recognition by the helix bundle domain and how this effect happens within the full length
- one publication on a novel alignment media that has been developed to obtained structural and dynamic information about the primase and the catalytic domain at high temperature
- one publication about how the catalytic domain interact with nucleotides and how it influences our understanding of the catalytic cycle of this system
The study of complex interaction involving a large protein (the primase) and several co-factors (DNA and multiple NTPs) is an extremely challenging study for NMR spectroscopy. In particular, those processes are transient, involving a complex pattern of interactions. Those interactions induce important conformational dynamical changes both at the local level and in the overall organization of the system. Developing approaches to study such complex biological system is clearly pushing the state-of-the-art limit of NMR spectroscopy, usually limited to simpler system in terms of size and number of compounds interacting. Those developments are long and challenging but very much needed to enable NMR spectroscopy to investigate real biological machineries in action.
In the course of this study, we have developed an active collaboration with Prof. Beat Meier at ETH Zurich to study the primase-DNA complex in presence and absence of NTPs using sold-state NMR. By combining our knowledge on this system and their expertise in solid-state NMR, we have started to investigate this system using 1H-detected ultra-fast magic angle spinning. This approach, that looks very promising, is also at the very edge of actual technological development and pushing towards novel strategies to characterize such system at atomic resolution.
Here, we have progressed toward the complete understanding of a primase catalytic cycle at atomic resolution. It is clear that additional work is needed to fully finish this research project. However the DNA replication initiation mechanism has never been unraveled at atomic resolution before. The novel results provided here allow to significantly increase our knowledge and understanding these complex processes.
The primase studied here is relatively close from eukaryotic and thus human primases. This makes on the long term a study potentially interesting for the development of drugs selectively targeting the DNA initiation mechanism. Of particular interest would be to investigate similar system in different organisms (eukaryotes, bacteria...) to find precise differences that can be exploited to develop e.g. novel antibiotics. The current state of research does not yet provide enough knowledge for rational drug design of the DNA primases but this study helps in moving towards this direction.
In the course of this study, we have developed an active collaboration with Prof. Beat Meier at ETH Zurich to study the primase-DNA complex in presence and absence of NTPs using sold-state NMR. By combining our knowledge on this system and their expertise in solid-state NMR, we have started to investigate this system using 1H-detected ultra-fast magic angle spinning. This approach, that looks very promising, is also at the very edge of actual technological development and pushing towards novel strategies to characterize such system at atomic resolution.
Here, we have progressed toward the complete understanding of a primase catalytic cycle at atomic resolution. It is clear that additional work is needed to fully finish this research project. However the DNA replication initiation mechanism has never been unraveled at atomic resolution before. The novel results provided here allow to significantly increase our knowledge and understanding these complex processes.
The primase studied here is relatively close from eukaryotic and thus human primases. This makes on the long term a study potentially interesting for the development of drugs selectively targeting the DNA initiation mechanism. Of particular interest would be to investigate similar system in different organisms (eukaryotes, bacteria...) to find precise differences that can be exploited to develop e.g. novel antibiotics. The current state of research does not yet provide enough knowledge for rational drug design of the DNA primases but this study helps in moving towards this direction.