Focus on numbers - characterizing protein accumulation at DNA double-strand breaks.

Unrepaired DNA double-strand breaks can lead to cell death or genomic rearrangements. The DNA damage response (DDR) is a complex signaling cascade in which a plethora of factors act to finely tune repair pathway choice. Several DDR proteins have been shown to accumulate at sites of DNA lesions in characteristic dot-like structures known as DNA repair foci. Changes in foci brightness, commonly expressed in arbitrary intensity units, are often used as readout for DNA repair dynamics. However, due in part to technical challenges, the stoichiometry, absolute number of proteins recruited to DDR foci, and their impact on the resolution of the break remain incompletely characterized. Here, we combine spatial intensity distribution analysis (SpIDA) and a custom foci detection algorithm into an easy-to-use pipeline that, starting from confocal images, allows quantitative description of protein accumulation in DNA repair foci. Moreover, by quantifying foci based on their molecular count, SpIDA overcomes the limitations of ambiguous intensity units, enabling stoichiometric quantification between repair factors and providing a unifying means for experimental comparisons.