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.