Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

Abstract

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (γ-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Spatiotemporal dynamics of homologous recombination repair at single collapsed replication forks

“…Our observations thus support a loading mechanism that does not require RAD51–BRCA2 interaction at repair foci in order to achieve RAD51 nucleofilament seeding. Elucidation of delayed BRCA2 recruitment contrasts other studies that have detected its recruitment to DSBs within 30 s 53 or the first half hour following damage 54 ; however, previous observations of sub-minute protein recruitment have generally not only relied on massive, heterogeneous damage induced using laser microirradiation in order to achieve high temporal resolution but also confounding the damage response pathways in play and limiting colocalization to confocal measurements 53 , 55 – 57 . Similarly, two-ended DSBs induced by ionizing radiation are not analogous to the HR-repaired seDSBs such as those we examine here and those that are predominantly caused by endogenous stress 4 , potentially resulting in different repair pathways.…”

SHLD 2/ FAM 35A co‐operates with REV 7 to coordinate DNA double‐strand break repair pathway choice

“…U2OS stable cell populations expressing the various constructs were transferred to a 96‐well plate with 170 μm glass bottom (Ibidi), pre‐sensitized with 10 μg/ml Hoescht 33342, and microirradiated using a FV‐3000 Olympus confocal microscope equipped with a 405 nm laser line as described previously (Gaudreau‐Lapierre et al , ). Immunofluorescence was performed as described previously (Gaudreau‐Lapierre et al , ). Briefly, following micro‐irradiation, cells were allowed to recover before pre‐extraction in 1× PBS containing 0.5% Triton X‐100 on ice for 5 min.…”

FAM35A co-operates with REV7 to coordinate DNA double-strand break repair pathway choice

“…U2OS stable cell populations expressing the various constructs were transferred to a 96well plate with 170 µm glass bottom (Ibidi), presensitized with 10 µg/mL Hoescht 33342 and microirradiated using a FV-3000 Olympus confocal microscope equipped with a 405nm laser line as described previously (Gaudreau-Lapierre et al, 2018). Immunofluorescence was performed as described previously (Gaudreau-Lapierre et al, 2018). Briefly, following microirradiation, cells were allowed to recover before pre-extraction in 1X PBS containing 0.5 % Triton X-100 on ice for five minutes.…”