Dissection of DNA damage and repair pathways in live cells by femtosecond laser microirradiation and free-electron modeling

作者：Michael Schmalz, Xiaoxuan Liang, Ines Wieser, Caroline Gruschel, Lukas Muskalla, Martin Stöckl, Roland Nitschke, Norbert Linz, Alfred Leitenstorfer, Alfred Vogel, Elisa Ferrando‐May · 发表于：Proceedings of the National Academy of Sciences · 年份：2023 · DOI：10.1073/pnas.2220132120 · 被引用次数：18 · 研究领域：DNA Repair Mechanisms、Photodynamic Therapy Research Studies、bioluminescence and chemiluminescence research

Understanding and predicting the outcome of the interaction of light with DNA has a significant impact on the study of DNA repair and radiotherapy. We report on a combination of femtosecond pulsed laser microirradiation at different wavelengths, quantitative imaging, and numerical modeling that yields a comprehensive picture of photon-mediated and free-electron-mediated DNA damage pathways in live cells. Laser irradiation was performed under highly standardized conditions at four wavelengths between 515 nm and 1,030 nm, enabling to study two-photon photochemical and free-electron-mediated DNA damage in situ. We quantitatively assessed cyclobutane pyrimidine dimer (CPD) and γH2AX-specific immunofluorescence signals to calibrate the damage threshold dose at these wavelengths and performed a comparative analysis of the recruitment of DNA repair factors xeroderma pigmentosum complementation group C (XPC) and Nijmegen breakage syndrome 1 (Nbs1). Our results show that two-photon-induced photochemical CPD generation dominates at 515 nm, while electron-mediated damage dominates at wavelengths ≥620 nm. The recruitment analysis revealed a cross talk between nucleotide excision and homologous recombination DNA repair pathways at 515 nm. Numerical simulations predicted electron densities and electron energy spectra, which govern the yield functions of a variety of direct electron-mediated DNA damage pathways and of indirect damage by • OH radicals resulting from laser and electron intera...