Periodic Reporting for period 3 - nanoDNArepair (Next Generation Nanofluidic Devices for Single Molecule Analysis of DNA Repair Dynamics)
Reporting period: 2023-04-01 to 2024-09-30
Double-strand breaks (DSBs) in chromosomes are one of the many types of DNA damage that can occur via normal cellular processes or environmental exposures. DSBs can lead to cell death if left unrepaired; if not repaired properly, they can cause deletions, translocations and fusions in the DNA. By its nature, a DSB is located at the end of a DNA strand, rendering it difficult to study with conventional single DNA molecule methods that involve anchoring the DNA at its ends to hold it in place. The EU-funded nanoDNArepair project is creating a nanofluidics platform to study DNA repair at a single-molecule level. After trapping the DNA within a nanofluidic channel, proteins can be added or removed through a slit perpendicular to the length of the channel. The pioneering setup will enable access to unprecedented spatio-temporal details of DNA repair processes on genomic length DNA on single DNA molecules.
Four papers using the nanofluidic devices that are at the core of the proposal have been published.
The first is a study where we use a newly developed nanofluidic device that allows studies of dynamic DNA-protein interactions. We demonstrated how we can study DNA compaction and de-compaction, induced by a polycationic protein, in real time. We can trap DNA without protein bound in the device, add protein, and subsequently remove protein by adding proteinase K and retrieve the original DNA conformation. We are now going to work on more complex designs so that we can add more than two different components and with higher precision and faster switching than in the current design.
The second study was on a chaperone protein from the HIV-virus. It is again an important proof-of-concept study that highlights many of the features of the nanofluidics method that make it suitable for the studies of repair of DNA double-strand breaks that are at the core of the proposal. We demonstrated that the protein forms local compactions on DNA and using the dynamic device we could show how these are formed. The most important observation in the light of this proposal was however that, also using the dynamic device, we demonstrated how a protein can link two DNA ends together. The DNA used has complementary overhangs and the protein can hybridize the ends to form a “repaired” DNA.
The two next stories are devoted to repair of double-strand breaks. The first is the first single DNA molecule study of Non-Homologous End-Joining (NHEJ) in bacteria. NHEJ in bacteria is an interesting process since it only involves two proteins, Ku and LigD, while the corresponding mammalian system is very complex with many partners. We combined our nanofluidics method with magnetic tweezers studies in the group of T. Strick at CNRS to study how the process works in detail. We demonstrated that Ku alone can bridge two DNA ends and that the stability is largely dependent on the number of bases on the single strand overhangs. The stability was further enhanced by binding of LigD via interactions with the C-terminal tail of Ku. Finally, we demonstrated that Ku remains bound when the repair process is completed, meaning that in living bacteria there must be another system at hand to remove the protein when repair is completed.
Regarding double-strand breaks our second published study is devoted to the human protein CtIP. CtIP is primarily known as a cofactor of the MRN-complex, one of the key players in the early steps of repair of double-strand breaks. However, inspired by a recent paper we were curious to study how CtIP interacts with DNA without MRN present. Using high-throughput imaging in the nanofluidic devices, and comparing the wild-type protein to several structural mutants, we could demonstrate that the wild-type protein, that in its native form is a tetramer, can bridge DNA molecules to two DNA ends to come in close proximity. We demonstrated that the tetramer-state is crucial for this activity but that the length of the linkers connecting the four head domains is less important. This might be a new role that CtIP plays that has not been considered before.
The first is a study where we use a newly developed nanofluidic device that allows studies of dynamic DNA-protein interactions. We demonstrated how we can study DNA compaction and de-compaction, induced by a polycationic protein, in real time. We can trap DNA without protein bound in the device, add protein, and subsequently remove protein by adding proteinase K and retrieve the original DNA conformation. We are now going to work on more complex designs so that we can add more than two different components and with higher precision and faster switching than in the current design.
The second study was on a chaperone protein from the HIV-virus. It is again an important proof-of-concept study that highlights many of the features of the nanofluidics method that make it suitable for the studies of repair of DNA double-strand breaks that are at the core of the proposal. We demonstrated that the protein forms local compactions on DNA and using the dynamic device we could show how these are formed. The most important observation in the light of this proposal was however that, also using the dynamic device, we demonstrated how a protein can link two DNA ends together. The DNA used has complementary overhangs and the protein can hybridize the ends to form a “repaired” DNA.
The two next stories are devoted to repair of double-strand breaks. The first is the first single DNA molecule study of Non-Homologous End-Joining (NHEJ) in bacteria. NHEJ in bacteria is an interesting process since it only involves two proteins, Ku and LigD, while the corresponding mammalian system is very complex with many partners. We combined our nanofluidics method with magnetic tweezers studies in the group of T. Strick at CNRS to study how the process works in detail. We demonstrated that Ku alone can bridge two DNA ends and that the stability is largely dependent on the number of bases on the single strand overhangs. The stability was further enhanced by binding of LigD via interactions with the C-terminal tail of Ku. Finally, we demonstrated that Ku remains bound when the repair process is completed, meaning that in living bacteria there must be another system at hand to remove the protein when repair is completed.
Regarding double-strand breaks our second published study is devoted to the human protein CtIP. CtIP is primarily known as a cofactor of the MRN-complex, one of the key players in the early steps of repair of double-strand breaks. However, inspired by a recent paper we were curious to study how CtIP interacts with DNA without MRN present. Using high-throughput imaging in the nanofluidic devices, and comparing the wild-type protein to several structural mutants, we could demonstrate that the wild-type protein, that in its native form is a tetramer, can bridge DNA molecules to two DNA ends to come in close proximity. We demonstrated that the tetramer-state is crucial for this activity but that the length of the linkers connecting the four head domains is less important. This might be a new role that CtIP plays that has not been considered before.
Our work is beyond state of the art from several aspects, both methodological and biological.
We are working on further developing the nanofluidics assay to allow complex dynamic reactions to be investigated. We are taking steps in this direction. We have observed reactions promoted by proteins where two DNA ends are bridged, which is the first step in DNA repair. We are working on showing this for repair proteins and we are expanding the capabilities of the dynamic nanofluidic device to study complex reactions.
We have also revealed new biological insights into repair of broken DNA. For bacteria the NHEJ reaction had not been investigated on the single DNA molecule level before. We revealed important details about the mechanics of DNA tethering and also proposed biological mechanisms that must be at play in order for NHEJ to be efficient. For CtIP in human cells we explored the interaction of CtIP with DNA on its own where we demonstrated that it can bridge DNA to force two DNA ends to come in close proximity, a crucial step in repair of broken DNA. The biological role of this is yet to be confirmed.
We are working on further developing the nanofluidics assay to allow complex dynamic reactions to be investigated. We are taking steps in this direction. We have observed reactions promoted by proteins where two DNA ends are bridged, which is the first step in DNA repair. We are working on showing this for repair proteins and we are expanding the capabilities of the dynamic nanofluidic device to study complex reactions.
We have also revealed new biological insights into repair of broken DNA. For bacteria the NHEJ reaction had not been investigated on the single DNA molecule level before. We revealed important details about the mechanics of DNA tethering and also proposed biological mechanisms that must be at play in order for NHEJ to be efficient. For CtIP in human cells we explored the interaction of CtIP with DNA on its own where we demonstrated that it can bridge DNA to force two DNA ends to come in close proximity, a crucial step in repair of broken DNA. The biological role of this is yet to be confirmed.