Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation

Abstract

In this study the femtosecond near-IR and nanosecond green lasers are used to induce alterations in mitotic chromosomes. The subsequent double-strand break responses are studied. We show that both lasers are capable of creating comparable chromosomal alterations and that a phase paling observed within 1–2 s of laser exposure is associated with an alteration of chromatin as confirmed by serial section electron microscopy, DAPI, γH2AX and phospho-H3 staining. Additionally, the accumulation of dark material observed using phase contrast light microscopy (indicative of a change in refractive index of the chromatin) ∼34 s post-laser exposure corresponds spatially to the accumulation of Nbs1, Ku and ubiquitin. This study demonstrates that chromosomes selectively altered in mitosis initiate the DNA damage response within 30 s and that the accumulation of proteins are visually represented by phase-dark material at the irradiation site, allowing us to determine the fate of the damage as cells enter G1. These results occur with two widely different laser systems, making this approach to study DNA damage responses in the mitotic phase generally available to many different labs. Additionally, we present a summary of most of the published laser studies on chromosomes in order to provide a general guide of the lasers and operating parameters used by other laboratories.

DNA Damage to a Single Chromosome End Delays Anaphase Onset

“…PtK1 cells stably expressing a green fluorescent protein (GFP)-tagged Nbs1 previously generated (27) were incubated with Advanced F-12/DMEM supplemented with L-glutamine, 4% FBS, and antibiotics. All cell types were incubated at 37°C with 5% CO 2 .…”

“…Laser Exposure and Dosimetry-A previously described custom RoboLase ablation system was used in these studies (27,33,40). Briefly, the system uses a femtosecond pulsed titaniumsapphire near-infrared (NIR) laser (Coherent Inc., Santa Clara, CA) coupled to a motorized inverted Zeiss microscope (Axiovert 200 M) with a 37°C culture dish stage (Warner Instruments, LLC).…”

“…Single chromosome tips (TIPs) and chromosome arms (non-TIPs) of live unsynchronized metaphase cells were microirradiated using the 200-fs pulse width NIR emission wavelength of 800 nm with a diffraction-limited (0.7-m diameter) focal spot as shown previously (33). Due to the ultrashort pulse duration, damage was confined to the focal spot (26,27). Individual laser exposure (irradiation) to single TIP and non-TIP of cells was performed at a dose range of 2.43-2.65 ϫ10 ϩ11 watts/cm 2 .…”

“…Laser microirradiation has been shown to produce predominantly DSBs, akin to ionizing radiation, eliciting the DDR in mammalian interphase cells, mitotic chromosomes, and anaphase telomeres of PtK2 cells (26 -33). Previous studies have shown that laser-induced DNA breaks on mitotic chromosomes do not result in chromosome fragmentation, and DSBs are only introduced at the laser focal spot (27,33). The spatio-temporal control of laser ablation permits the generation of damaged chromosome tips without genetic perturbation or long term mitotic arrest.…”

Imaging of the DNA damage‐induced dynamics of nuclear proteins via nonlinear photoperturbation

“…The radiation employed for this purpose ranges from X-rays [6] to charged particles [7] to laser emission at various wavelengths [8][9][10][11]. For proteins that are attracted to DNA lesions, as e.g.…”