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

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.

Simultaneous dual-channel imaging to quantify interdependent protein recruitment to laser-induced DNA damage sites

“…The type and the amount of DNA damage induced by laser microirradiation is known to depend on the laser type, intensity and the use of presensitizers [3,5,11,12]. U2OS cells were presensitized with BrdU and 355 nm UV-A laser was applied in two settings: (i) at 20% laser intensity (130 μW at the sample) and laser pulse duration of 5 ms/pixel (for recruitment analysis shown in Figure 2–6) and (ii) at 80% laser intensity (820 μW at the sample) and laser pulse duration of 0.3 ms/pixel (for recruitment analysis shown in Figure 7).…”

“…Variable efficiency of BrdU incorporation may result in a variable density of strand breaks, which is known to affect the kinetics of protein recruitment to DNA damage sites [3]. …”

“…DNA lesions are usually induced as spots or stripes of laser-microirradiated regions across a nucleus. Damage can be induced using in-built lasers (e.g., 405 nm lasers targeted to cells in the FRAP mode of laser-scanning confocal microscopes) or add-on laser modules, such as UV lasers and near infrared (800 nm) multiphoton lasers [3]. Induction of DNA damage using low-energy UV-A lasers (315–400 nm) is facilitated by presensitization with DNA-intercalating Hoechst 33258 and 33342 dyes or with a nucleoside analogue 5-bromo-2ʹ-deoxyuridine (BrdU), which obviate the need for high laser intensity [1,4].…”

“…UV-A laser light typically generates UV-type lesions such as cyclobutane pyrimidine dimers (CPDs) and 6–4 photoproducts (6–4PPs), as well as oxidative base damage and abasic sites due to reactive oxygen species produced in the aqueous cellular environment [7,8]. UV-A laser is thus particularly suitable for studying base excision repair (BER) [3]. Presensitization facilitates single-strand breaks (SSBs) and double-strand breaks (DSBs) upon UV activation, and thus enables the study of proteins involved in single-strand break repair (SSBR) and double-strand break repair (DSBR) as well [1–4,9].…”

“…Kinetics of protein recruitment to laser-induced DNA damage sites may vary depending on the laser type, laser power and presensitization methods (BrdU, Hoechst, or both), all of which influence the type of damage that is generated within DNA, as well as the type of cell line (most commonly used are U2OS and HeLa), fluorescent tags and protein expression levels [3,5,11,12]. Comparative analysis of recruitment kinetics revealed a general agreement between the temporal protein recruitment sequence and the timing of the respective step in DNA damage response in which the protein exerts its function; DNA damage-sensing proteins are recruited first, followed by proteins involved in early steps of DSB repair and chromatin remodelers, whereas proteins involved in DNA damage signalling and homologous recombination exhibit a broader range of recruitment timing [8,12].…”