DNA Repair Pathways

DNA Repair Pathways

The DNA damage in cultured living cells was induced by localized 266 nm radiation (sub-nuclear damage induction). The cells expressed GFP tagged repair factors GFP-XPA and Rad54-GFP to follow the nucleotide excision repair (NER) and double strand break repair (DSB) mechanisms, respectively.
Experimental information: 

Imaging: Zeiss Axiovert 200M microscope modified for UV transmission equipped with LSM510 confocal module

DNA damage: 2 mW 266 nm pulsed (7.8 kHz) DPSS laser (DPSL-266) and fixed spot illumination system (AiWon) 

(A) GFP-XPA expressing cells were irradiated with 266 nm either without (arrow) or with attenuation (arrowhead). GFP-XPA accumulates on both areas (green, left panel) whereas TUNEL (red, middle panel) only stains positive on the spot that was created without attenuation. (B) GFP-XPA expressing cells were irradiated by attenuated UV-C laser light (arrow). Presence of CPDs was shown by immunohistochemical staining with -CPD
(red, middle panel). (C) Cells that were irradiated in G1 or G2 phase (homogeneous PCNA pattern, red, middle panel) show no accumulation of Rad54-GFP (green, left panel) 2 hours after irradiation (arrow). (D) In cells that were irradiated in S phase (PCNA pattern in foci, red, middle panel) Rad54-GFP (green, left panel) accumulates at locally irradiated areas within 1 hour after irradiation (arrow).

Data from: Dinant et al. (2007) Activation of multiple DNA Repair Pathways by Sub-nuclear Damage Induction Methods, J. Cel. Sci. 120, 2731-40.

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