Comparative analysis of different laser systems to study cellular responses to DNA damage in mammalian cells - PubMed (original) (raw)

Comparative Study

Comparative analysis of different laser systems to study cellular responses to DNA damage in mammalian cells

Xiangduo Kong et al. Nucleic Acids Res. 2009 May.

Abstract

Proper recognition and repair of DNA damage is critical for the cell to protect its genomic integrity. Laser microirradiation ranging in wavelength from ultraviolet A (UVA) to near-infrared (NIR) can be used to induce damage in a defined region in the cell nucleus, representing an innovative technology to effectively analyze the in vivo DNA double-strand break (DSB) damage recognition process in mammalian cells. However, the damage-inducing characteristics of the different laser systems have not been fully investigated. Here we compare the nanosecond nitrogen 337 nm UVA laser with and without bromodeoxyuridine (BrdU), the nanosecond and picosecond 532 nm green second-harmonic Nd:YAG, and the femtosecond NIR 800 nm Ti:sapphire laser with regard to the type(s) of damage and corresponding cellular responses. Crosslinking damage (without significant nucleotide excision repair factor recruitment) and single-strand breaks (with corresponding repair factor recruitment) were common among all three wavelengths. Interestingly, UVA without BrdU uniquely produced base damage and aberrant DSB responses. Furthermore, the total energy required for the threshold H2AX phosphorylation induction was found to vary between the individual laser systems. The results indicate the involvement of different damage mechanisms dictated by wavelength and pulse duration. The advantages and disadvantages of each system are discussed.

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Figures

Figure 1.

Figure 1.

Induction of different types of DNA damage by UVA, ns and ps green, and NIR lasers. At 3–5 min after damage induction by the different lasers indicated at the top, cells were fixed and stained with antibodies specific for CPD, 6-4PP and 8-oxoG. Corresponding brightfield phase contrast images are also shown. Scale bar = 5 µm.

Figure 2.

Figure 2.

Recruitment of SSB repair and BER factors to the damage sites induced by UVA, green and NIR lasers. Immediately following the damage induction, cells were fixed and stained with antibodies specific for PARP-1, XRCC1 or FEN-1. The location of the induced lesions are indicated by arrowheads. Corresponding brightfield phase contrast images are also shown. Scale bar = 5 µm.

Figure 3.

Figure 3.

DSB responses induced by different laser systems. (A) Ku recruitment to the laser-induced damage sites. Immunostaining of cells damaged by different lasers as indicated using anti-Ku antibody. Lesions are indicated by arrowheads. Corresponding brightfield phase contrast images are also shown. Scale bar = 5 µm. (B) 53BP1 accumulation, hSMC1 phosphorylation and cohesin accumulation at the damage sites. Cells damaged by different lasers as indicated were fixed and stained with antibodies specific for 53BP1, phosphorylated hSMC1 (S966P), or the non-SMC cohesin subunit SA2. Lesions are indicated by arrowheads. Corresponding brightfield phase contrast images for SA2 staining are also shown. Scale bar = 5 µm.

Figure 4.

Figure 4.

Mechanisms of DNA damage by different laser microbeam systems. Three possible damage mechanisms (single-photon absorption, multi-photon absorption, and plasma formation) and their associated thermal, chemical and mechanical effects are listed. Based on the γH2AX-threshold and working laser parameters (e.g. wavelength, pulse duration and frequency, peak irradiance and total input energy), the most likely mechanisms of damage induction by UVA, UVA with BrdU, cw blue, cw blue with BrdU, ns and ps green, and fs NIR are indicated. Although the thermal effect is most likely an important mechanism of damage induction, it is highly restricted temporally and spatially, and is dispersed instantaneously, and therefore any heat-inflicted damage outside of the focused area or any temperature rise in the cell is not expected to occur under the conditions used. Further study is necessary to delineate which mechanisms and/or combination of mechanisms impact each of the laser and biological systems discussed.

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