Real-time studies of DNA repair kinetics following low-LET short-pulse electron radiation

Radiation-induced damage to the genomic DNA of cells may lead to errors in transcription and replication and, if not repaired correctly, these may result in mutations, genomic instability and cell death. Laser microbeams have generally been used by many research groups to investigate the real-time dynamics of protein recruitment in response to DNA insults in mammalian cells; however, such irradiations induce a plethora of DNA damage (including UV base damage, base damage, SSBs and DSBs and complex damage). A novel experimental setup has been designed capable of following the dynamics of protein recruitment in response to DNA insults in mammalian cells shortly following submicrosecond- pulsed electron irradiation of living mammalian cells, not possible using conventional irradiation techniques. This arrangement was developed based on a 6 MeV electron pulse linear accelerator, to deliver sparsely ionising radiation, coupled to an automated, time-lapse inverted epifluorescence microscope imaging system. An integrated robotic system contained within a physiological environment of 37°C and 5% CO2 was used to transfer remotely and repetitively custom-designed cell dishes containing the mammalian cells between irradiation and imaging locations.
Following the development of the linear accelerator and associated imaging devices, preliminary ‘proof-of-principle’ investigations were carried out using living HT1080 mammalian cells containing YFP-tagged 53BP1, an established biomarker of DSB, to follow the recruitment and loss of 53BP1 to sites of radiation-induced DNA damage in real-time. This novel experimental setup has allowed for the first time observations of the appearance and disappearance of radiation-induced foci in the same cell population at very early times. These single-foci studies have provided evidence for the formation of not only promptly formed DSBs but also late appearing DNA damage signalled by 53BP1. These data highlight different classes of DSBs formed in response radiation damage.
Additionally, the role of cell cycle on the repair kinetics was undertaken using HT1080- 53BP1-YFP cells which also express Geminin-mCherry under appropriate selection. Geminin is increasingly expressed from early S-phase onwards, but is degraded following mitosis. Geminin-associated fluorescence can be used as a marker of progression through the cell cycle. 53BP1 repair kinetics were characterised in response to radiation damage in combination with ATM and PARP inhibitors. These studies provided supporting evidence for the existence of different classes of DSBs, potentially assigned to radiation-induced replication breaks and DSBs formed by enzymatic conversion of clustered damage.
These preliminary ‘proof-of-principle’ findings using DNA damage repair as an example, emphasize the use of this novel technology to explore the dynamics of numerous other biochemical processes in living cells in real-time with the knowledge of being able to quantify the range of damage induced by IR coupled with accurate dosimetry. The knowledge obtained may be used to identify potential biological targets coupled with drug discovery for translation into adjuncts for radiotherapy.

Keywords: DNA repair
Radiobiology
DNA damage
53BP1
Cancer
DNA repair kinetics
Linac
Real-time DNA repair kinetics
Oncology
LET
Radiation Biology
Advisor: O'Neill, P
Vojnovic, B