Regensburg 2010 – scientific programme
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BP: Fachverband Biologische Physik
BP 6: Posters: DNA and DNA Enzymes
BP 6.7: Poster
Monday, March 22, 2010, 17:15–20:00, Poster B1
Visualisation of PCNA monoubiquitination in vivo by single pass spectral imaging FRET microscopy — •Christopher Batters1, Hannah Zhu2, and Julian Sale2 — 1Institute of Physiology, Ludwig-Maximilians-Universität, Pettenkofestr. 12, 80336 München, Germany — 2Medical Research Council Laboratory of Molecular Biology, Division of Protein & Nucleic Acid Chemistry, Hills Road, Cambridge, CB2 0QH, U.K.
Monoubiquitination of the DNA sliding clamp, PCNA, plays a central role in the control of damage bypass during replication. By combining a widely-spaced FRET donor/acceptor pair (CFP and mRFP) with spectral imaging, we have developed a simple method for the visualisation of PCNA monoubiquitination in both fixed and live cells with a single imaging pass. We validate the method with genetic controls in the avian cell line DT40 and examine the intracellular dynamics of PCNA ubiquitination following subnuclear UV irradiation. This general approach is likely to be of utility for live imaging of monoubiquitination and sumoylation of a wide range of substrates in vivo.