Systematic analysis of DNA damage induction and DNA repair pathway activation by continuous wave visible light laser micro-irradiation

Abstract

Laser micro-irradiation can be used to induce DNA damage with high spatial and temporal resolution, representing a powerful tool to analyze DNA repair in vivo in the context of chromatin. However, most lasers induce a mixture of DNA damage leading to the activation of multiple DNA repair pathways and making it impossible to study individual repair processes. Hence, we aimed to establish and validate micro-irradiation conditions together with inhibition of several key proteins to discriminate different types of DNA damage and repair pathways using lasers commonly available in confocal microscopes. Using time-lapse analysis of cells expressing fluorescently tagged repair proteins and also validation of the DNA damage generated by micro-irradiation using several key damage markers, we show that irradiation with a 405 nm continuous wave laser lead to the activation of all repair pathways even in the absence of exogenous sensitization. In contrast, we found that irradiation with 488 nm laser lead to the selective activation of non-processive short-patch base excision and single strand break repair, which were further validated by PARP inhibition and metoxyamine treatment. We conclude that these low energy conditions discriminated against processive long-patch base excision repair, nucleotide excision repair as well as double strand break repair pathways.

Keywords: DNA damage; DNA repair; laser micro-irradiation; live-cell microscopy; processive DNA synthesis.

Figure 1.. Laser micro-irradiation with 405 nm induces DSBs and activates homologous recombination and non-homologous end joining without exogenous sensitizers.

HeLa Kyoto cells were micro-irradiated with 405, 488 or 561 nm laser lines at 1, 3, and 8 mJ respectively, fixed immediately (5 min lag time) and subsequently stained for the different DNA damage markers and DNA counterstained with DAPI. Arrows indicate the sites of micro-irradiation. Scale bars = 10 µm. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (A) and immunofluorescence staining against the double strand break marker γH2AX (B) exclusively marks sites of 405 nm induced DNA damage and not after 488 or 561 nm. (C) Schematic representation of homologous recombination (HR) and non-homologous end joining (NHEJ), with analyzed proteins highlighted in green. (D) HeLa Kyoto cells, stably expressing mCherry-PCNA, were transfected with GFP-Rad51 (D) or GFP-Ligase 4 (F) and then irradiated with different laser lines as indicated at 1, 3, and 8 mJ for the 405, 488 and 561 nm lines respectively. Representative confocal microscopy images are shown from micro-irradiation experiments with 405 nm laser (D and F). Arrowheads point to the sites of micro-irradiation, shown enlarged as insets. Scale bars = 5 µm. (E and G) The plots show the accumulation of the different repair proteins over time as mean value. Error bars represent standard deviation. (H) Bar histograms display the calculated mean maximal accumulation for the different repair factors as indicated. Error bars represent standard deviation.

Figure 2.. Micro-irradiation with 405 but not with 488 and 561 nm lasers induces DNA photo-products and activates nucleotide excision repair.

(A) Immunofluorescent detection of cyclobutane dimers show a signal at the sites of micro-irradiation with 405 nm laser but no signal after 488 or 561 nm irradiation in HeLa cells. (B) Simplified schematics for the NER pathway with analyzed proteins highlighted in green. (C) HeLa Kyoto cells, stably expressing mCherry-PCNA transfected with XPC-GFP were micro-irradiated at the indicated spots and analyzed by time-lapse microscopy. Scale bar = 5 µm. (D) Measured mean accumulation of XPC-GFP after micro-irradiation with the indicated wavelength. Curves represent means and error bars are standard deviation. (E) Bar histograms display the calculated mean maximal accumulation for the different repair factors and wavelengths as indicated. Error bars represent standard deviation.

Figure 3.. Discrimination against processive DNA repair with 405 versus 488 and 561 nm micro-irradiation.

(A) Schematic representation of short- and long-patch base excision repair. (B) HeLa Kyoto cells stably expressing mCherry-PCNA were transfected with GFP-XRCC1 and irradiated with 405, 488 and 561 nm laser set to 1, 3, and 8 mJ respectively. Arrowheads point to the sites of micro-irradiation, shown enlarged as insets. Scale bars: 5 µm. The plots represent the accumulation of the different repair proteins over time as mean value. Error bars represent standard deviation. (C) Bar histograms display the calculated mean maximal accumulation for the different repair factors as indicated. Error bars represent standard deviation. (D) Different accumulation kinetics for XRCC1 after micro-irradiation with lasers of different wavelength- Lines present the mean values and error bars are calculated as standard deviations. (E) Half-times till maximum accumulation of XRCC1 after micro-irradiation with 405, 488 and 561 nm laser set to 1, 3, and 8 mJ respectively. Bars present means and error bars are standard deviations.

Figure 4.. Micro-irradiation in PARP inhibited or methoxyamine treated cells.

Time lapse imaging of HeLa Kyoto cells stably expressing mCherry-PCNA and transfected with GFP-XRCC1, either mock treated (A) or pretreated with Olaparib (B) and micro-irradiated with 405 nm. Lower panels shows the mean accumulation and error bars represent standard deviation. (C) Quantification of wavelength dependent accumulation of PCNA and XRCC1 in mock or Olaparib treated cells. Bars represent the mean maximum accumulation and error bars indicate the standard deviation. (D) Quantification of micro-irradiation induced maximum accumulation in cells not exposed (upper panel) or exposed to methoxyamine. Bars represent the means and error bars are standard deviation. The statistical significance of methoxyamine treatment was tested by a two-tailed Mann-Whitney-U test (95% confidence interval) between the treated and untreated sample and is indicated as *: p ≤ 0.05; **: p ≤ 0.01 and ns: p > 0.05.