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

Abstract

Fluorescence microscopy in combination with the induction of localized DNA damage using focused light beams has played a major role in the study of protein recruitment kinetics to DNA damage sites in recent years. Currently published methods are dedicated to the study of single fluorophore/single protein kinetics. However, these methods may be limited when studying the relative recruitment dynamics between two or more proteins due to cell-to-cell variability in gene expression and recruitment kinetics, and are not suitable for comparative analysis of fast-recruiting proteins. To tackle these limitations, we have established a time-lapse fluorescence microscopy method based on simultaneous dual-channel acquisition following UV-A-induced local DNA damage coupled with a standardized image and recruitment analysis workflow. Simultaneous acquisition is achieved by spectrally splitting the emitted light into two light paths, which are simultaneously imaged on two halves of the same camera chip. To validate this method, we studied the recruitment of poly(ADP-ribose) polymerase 1 (PARP1), poly (ADP-ribose) glycohydrolase (PARG), proliferating cell nuclear antigen (PCNA) and the chromatin remodeler ALC1. In accordance with the published data based on single fluorophore imaging, simultaneous dual-channel imaging revealed that PARP1 regulates fast recruitment and dissociation of PARG and that in PARP1-depleted cells PARG and PCNA are recruited with comparable kinetics. This approach is particularly advantageous for analyzing the recruitment sequence of fast-recruiting proteins such as PARP1 and ALC1, and revealed that PARP1 is recruited faster than ALC1. Split-view imaging can be incorporated into any laser microirradiation-adapted microscopy setup together with a recruitment-dedicated image analysis package.

Direct measurement of protein–protein interactions by FLIM-FRET at UV laser-induced DNA damage sites in living cells

“…For example, laser microirradiation coupled with wide-field or confocal fluorescence microscopy has been broadly used for studying protein recruitment to the sites of laser-induced DNA damage ( 1–6 ). The efforts over the past two decades have yielded an increasingly detailed picture of the sequence in which DNA damage sensing factors, chromatin remodellers, and signalling and repair factors are recruited to and released from laser microirradiation sites ( 7–11 ). Recently, we developed a dual-channel simultaneous imaging approach to monitor and compare the recruitment kinetics of two proteins in response to DNA damage in the same cell ( 7 ).…”

“…Comparing the recruitment kinetics of proteins, however, does not allow conclusions about protein–protein interactions that facilitate their recruitment to DNA damage sites. Many DDR proteins are recruited to DNA damage sites in a poly(ADP-ribose) (PAR)-dependent manner ( 7 , 12–17 ). PAR is a post-translational protein modification generated primarily by poly(ADP-ribose) polymerase 1 (PARP1) upon binding to DNA strand breaks ( 18 ).…”

“…PARP1 activation at DNA damage sites triggers chromatin relaxation due to rapid PARylation of histones leading to histone displacement ( 19–21 ). Additionally, PARylation triggers a wave of PAR-dependent recruitment of proteins to the site of DNA damage ( 7 , 12–17 ). Some proteins such as the chromatin remodeller amplified in liver cancer 1 (ALC1) and the single-strand break (SSB) repair scaffold protein X-ray repair cross-complementing protein 1 (XRCC1) were shown to directly bind PAR and interact with PARP1 in response to DNA damage ( 7 , 12 , 13 , 17 , 22 , 23 ).…”

“…Additionally, PARylation triggers a wave of PAR-dependent recruitment of proteins to the site of DNA damage ( 7 , 12–17 ). Some proteins such as the chromatin remodeller amplified in liver cancer 1 (ALC1) and the single-strand break (SSB) repair scaffold protein X-ray repair cross-complementing protein 1 (XRCC1) were shown to directly bind PAR and interact with PARP1 in response to DNA damage ( 7 , 12 , 13 , 17 , 22 , 23 ). Unlike ALC1 and XRCC1, the chromatin remodellers CHD3 and CHD4 do not bind PAR and do not interact with PARP1 directly but require PAR-dependent chromatin relaxation for efficient recruitment to DNA damage sites ( 15 , 24 , 25 ).…”

In Situ Analysis of DNA-Protein Complex Formation upon Radiation-Induced DNA Damage

“…Of particular interest are those studies in which quantitative determination of the recruitment and release kinetics at the DNA damage sites was performed for direct players and regulators of the various DNA repair processes. Specific parameters were estimated during NER, (i.e., XPA, XPC, PCNA, p21) [67,68,69,70], in the base excision repair (BER) process (i.e., XRCC1, OGG1, PARP-1) [71,72,73,74,75,76,77], as well as in the non-homologous end joining (NHEJ) process (i.e., Ku70/80 complex, DNA-PK, DNA Ligase III) [78,79,80]. Furthermore, calculation of several kinetic parameters, such as the ratio between protein-bound state and diffusive state over time, allowed the further deepening of theoretical models regarding DNA–protein, or protein–protein complex formation upon DNA damage, and the evaluation quantitatively of the spatiotemporal activity of DNA repair factors [67,69,71,75].…”