Purification and Characterization of DNA Repair Proteins

Whole cell extracts (WCE) were prepared as previously described (26 (link),27 (link)). WCE from 20 g HeLa cell pellets (Cilbiotech, Belgium) was fractionated using column chromatography, and proteins present in active fractions from the final Mono-Q chromatography were identified by tandem mass spectrometry, as recently described (27 (link),28 (link)). Following each chromatography stage, protein fractions were analysed for in vitro BER activity using a free or mononucleosome substrate and active fractions pooled for the next chromatography step. Immunoblotting was performed as described in the references above, using the Odyssey Image Analysis System for protein detection and quantification. Primary antibodies raised against APE1 were kindly provided by Prof. G.Dianov, HECTD1 and Mule antibodies were from Bethyl Laboratories (Montgomery, USA), tubulin antibodies were from Sigma (Dorset, UK), Cul4A, DDB1 and histone H2B and H4 antibodies were from Abcam (Cambridge, UK), and histone H3 and H4 antibodies were from Cell Signaling (London, UK).