Spatiotemporal Dynamics of Early DNA Damage Response Proteins on Complex DNA Lesions

Abstract

The response of cells to ionizing radiation-induced DNA double-strand breaks (DSB) is determined by the activation of multiple pathways aimed at repairing the injury and maintaining genomic integrity. Densely ionizing radiation induces complex damage consisting of different types of DNA lesions in close proximity that are difficult to repair and may promote carcinogenesis. Little is known about the dynamic behavior of repair proteins on complex lesions. In this study we use live-cell imaging for the spatio-temporal characterization of early protein interactions at damage sites of increasing complexity. Beamline microscopy was used to image living cells expressing fluorescently-tagged proteins during and immediately after charged particle irradiation to reveal protein accumulation at damaged sites in real time. Information on the mobility and binding rates of the recruited proteins was obtained from fluorescence recovery after photobleaching (FRAP). Recruitment of the DNA damage sensor protein NBS1 accelerates with increasing lesion density and saturates at very high damage levels. FRAP measurements revealed two different binding modalities of NBS1 to damage sites and a direct impact of lesion complexity on the binding. Faster recruitment with increasing lesion complexity was also observed for the mediator MDC1, but mobility was limited at very high damage densities due to nuclear-wide binding. We constructed a minimal computer model of the initial response to DSB based on known protein interactions only. By fitting all measured data using the same set of parameters, we can reproduce the experimentally characterized steps of the DNA damage response over a wide range of damage densities. The model suggests that the influence of increasing lesion density accelerating NBS1 recruitment is only dependent on the different binding modes of NBS1, directly to DSB and to the surrounding chromatin via MDC1. This elucidates an impact of damage clustering on repair without the need of invoking extra processing steps.

An integrated on-line irradiation and in situ live cell imaging system

“…Analogous results have been reported by other groups using different cell lines and different indicating proteins [16,17]. It is interesting that DDR after c and a irradiation share the similar molecular basis and pathways while deviate substantially in the reaction time.…”

Nuclear dynamics of radiation-induced foci in euchromatin and heterochromatin

“…Radical scavengers are important factors to consider because 58% of low-LET (Linear Energy Transfer) ionizing radiation damage is generated by radicals [38]. However, a similar exclusion of RIF from heterochromatin occurs also when radical-induced DSBs are a minority, such as after exposure to densely ionizing particles, suggesting that other mechanisms are involved [13, 39, 40]. As we will discuss in this review, one main factor responsible for this non-uniform distribution is likely to be the dynamic relocalization of heterochromatic RIF to the euchromatic space during repair.…”

Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling

“…However, it is only applicable if the size of the cluster (the damaged area) is smaller than the bleached/photoactivated region. This model has been recently applied to describe the behavior of Nbs1 and Mdc1 at sites of DNA damage introduced by charged particle irradiation (Sprague et al, 2006; Tobias et al, 2013). …”