Technical Reference #1856

1856.

Stochastic and reversible assembly of a multiprotein DNA repair complex ensures accurate target site recognition and efficient repair Martijn S. Luijsterburg; Gesa von Bornstaedt; Audrey M. Gourdin; Antonio Z. Politi; Martijn J. Moné; Daniël O. Warmerdam; Joachim Goedhart; Wim Vermeulen; Roel van Driel; and Thomas Höfer, University of Amsterdam, The Journal of Cell Biology, 189(1856), (2010)
Link To Paper

Materials & Methods:
Cells were grown in glass-bottom dishes (MatTek) and locally UV irradiated as described previously (Moné et al. 2004; Luijsterburg et al. 2007). Individual cells were subsequently monitored for up to 6 h. Accumulation of EGFP-tagged repair proteins after local irradiation was quantified with Objective Image software. Time courses were normalized with respect to the plateau level. Start of the UV irradiation was defined as t = 0. The bound fraction of EGFP-tagged NER proteins in the local damage was calculated by the following equation: bound percent = (Ispot 􀀒 Ioutspot) × pixelsspot/(Inucleus 􀀒 Ibackground) × pixelsnucleus; where Ispot and Ioutspot are the mean pixel intensities inside the damaged spot and outside the spot respectively. Inucleus is the mean pixel intensity of the nucleus including the spot and Ibackground is the mean pixel intensity outside of the cell.

Microscopic Technique
Fluorescence microscope

Cell Type(s)
Human fibroblasts