Abstract
The DNA molecule is exposed to a multitude of damaging agents that can compromise its integrity: single (SSB) and double strand breaks (DSB), intra- or inter-strand crosslinks, base loss or modification, etc. Many different DNA repair pathways coexist in the cell to ensure the stability of the DNA molecule. The nature of the DNA lesion will determine which set of proteins are needed to reconstitute the intact double stranded DNA molecule. Multiple and sequential enzymatic activities are required and the proteins responsible for those activities not only need to find the lesion to be repaired among the millions and millions of intact base pairs that form the genomic DNA but their activities have to be orchestrated to avoid the accumulation of toxic repair intermediates. For example, in the repair of Single Strand Breaks (SSB) the proteins PARP1, XRCC1, Polymerase Beta and Ligase III will be required and their activities coordinated to ensure the correct repair of the damage.Furthermore, the DNA is not free in the nucleus but organized in the chromatin with different compaction levels. DNA repair proteins have therefore to deal with this nuclear organization to ensure an efficient DNA repair. A way to study the distribution of DNA repair proteins in the nucleus after damage induction is the use of the laser microirradiation with which a particular type of DNA damage can be induced in a localized region of the cell nucleus. The wavelength and the intensity of the laser used will determine the predominant type of damage that is induced. It is important to note that other lesions can also be generated at the microirradiated site. Living cells transfected with the fluorescent protein XRCC1-GFP are micro-irradiated under a confocal microscope and the kinetics of recruitment of the fluorescent protein is followed during 1 min. In our protocol the 405 nm laser is used to induce SSB.
Keywords: DNA damage, Single-strand DNA breaks, DNA repair, Microirradiation, Live imaging
Materials and Reagents
Equipment
Software
Procedure
Analysis and quantification
Representative data
Figure 1. HeLa cell expressing the XRCC1-GFP or the mutant protein XRCC1(L360D) were microirradiated with a 405 nm laser. A) Presented images correspond to the cell before microirradiation and 1 min after microirradiation. The microirradiation region is indicated by a dashed line. The mutant protein XRCC1(L360D), that has not any more the ability to be recruited to SSB was used as a control. B) Fluorescence intensity corresponding to XRCC1-GFP at the microirradiated region was quantified and displayed in the graph. Error bars represent the SEM of 10 independent cells.
Acknowledgments
We thank the IRCM microscopy facility. This work was funded by INSERM and grants from the Association pour la Recherche sur le Cancer (PJA 20131200165) and the CEA Radiobiology program.
References
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