DNA repair capability is one of the key indicators reflecting the biological consequences of cellular exposure to genotoxic insults. Determining the biochemical nature of the DNA damages induced by HZE particles and identifying the factors that are required for the repairing of these DNA lesions will provide tools to assess individual radiation susceptibility and also to validate risk assessment for human exposure to HZE particles. The goals of this study were two fold: 1) Develop new approaches to determine the types and spatial relationship among different DNA lesions formed along the dense ionizing tracks induced by HZE particles in human cells and to dissect the cellular capability to repair these DNA lesions in situ; and 2) Identify molecular pathways or factors that may be able to help the human cell respond efficiently to HZE particle-induced DNA lesions. For this purpose, investigators tested their hypothesis that specific nucleases, Artemis and/or Werner Syndrome protein (WRN), contribute specifically to the repair of complex DNA lesions induced by HZE particles. Investigators also aimed to determine the role of homologous recombination factors in repair of HZE particle-induced double strand breaks (DSBs). With these new approaches on hand, the investigators sought to provide a semi-quantitative measurement for the generation and spatial distribution of clustered DNA lesions along the dense track induced by HZE particles and determine the relative contribution of these Nonhomologous end-joining and homologous end joining factors in the repair of DNA DSBs, but also able to identify whether these repair activities play any role in managing clustered DNA lesions generated by HZE.
Experiment 1: Establishment of a base damage marker for live cell imaging and evaluation of kinetics of clustered DNA lesions in live cells.
- Identify a suitable base damage marker for live cell imaging.
- Evaluate the kinetics of recruitment of different DNA repair factors to the sites of DNA lesions induced by HZE particles in live cells.
Experiment 2: Establish approaches for cell synchronization and study induction and repair of clustered DNA lesions at different phases of cell cycle.
- Synchronize cells at different phases of cell cycle.
- Identify different phases of cell cycle.
- Study induction and repair of clustered DNA lesions at different phases of cell cycle.
- Determine cell cycle-dependent recruitment and retention of DNA repair proteins at the sites of clustered DNA lesions.
- Determine whether there is a differential repair capability of clustered DNA lesions in G1 vs. S phase of the human HT1080 cells.
- Monitor clustered DNA lesions repair in G2 phase of the cell cycle.
Experiment #3: Quantification and imaging analysis of clustered DNA lesions to determine whether the nature of DNA lesions induced by HZE particles is dependent on the physical properties of the HZE particles.
- Quantify charge and LET dependent induction and repair of clustered DNA lesions.
- Determine the nature of clustered DNA lesions induced by different HZE particles.
Homologous repair deficient hamster cells are highly sensitive to iron particles as compared with wild-type cells. WRN-deficient human cells are extremely sensitive to Fe particles as compared with gamma-rays. As DSBs induced by HZE particles are complex, and WRN has shown to have multiple roles in NHEJ, HR and BER pathways, it might play one or more roles in HZE particle induced DNA repair either by coordinating within the individual DNA repair pathways or it may coordinate between them. This could be achieved either by binding directly to the damaged DNA or recruited by other DNA-repair factors.
Artemis-deficient human cells are significantly sensitive to iron particles as compared with wild-type cells. However, the mechanism by which Artemis participates in the processing of complex DNA lesions is not clear. Artemis might be needed to process damaged ends at DSBs prior to rejoining. This might require DNA-PK- and ATM-dependent phosphorylation of Artemis followed by its activity modification from exonuclease to endonuclease.
FANCA-deficient human cells are highly sensitive to Fe particles as compared with FANCA deficient cells complemented with WT FANCA, suggesting a role for FA complex in the repair of clustered DNA lesions. However, whether all the factors of the FA complex orchestrate the repair of complex DNA lesions and the method by which these factors function in this processes is totally unknown.
No datasets exist for this study. A final report was archived.