Reconstitution of 3′-PG processing and subsequent gap filling in SCAN1 extracts by recombinant TDP1

<p><b>Copyright information:</b></p><p>Taken from "Deficiency in 3′-phosphoglycolate processing in human cells with a hereditary mutation in tyrosyl-DNA phosphodiesterase (TDP1)"</p><p>Nucleic Acids Research 2005;33(1):289-297.</p><p>Published online 12 Jan 2005</p><p>PMCID:PMC546157.</p><p>© 2005, the authors © </p> The plasmid substrate was treated with 3 mg/ml SCAN1 whole-cell extract (subject 1635) and analyzed as in . In some cases, 25 ng of recombinant TDP1 and/or 200 ng of XRCC4/DNA ligase IV complex (Trevigen) were added. Some samples contained 5 mM EDTA instead of 0.5 mM MgCl, as indicated

Processing of a 3′-PG 14mer by extracts of lymphoblastoid cells from SCAN1 patients (closed symbols) and from unaffected members (open symbols) of the same family, including TDP1+/– carriers (squares, males; circles, females)

<p><b>Copyright information:</b></p><p>Taken from "Deficiency in 3′-phosphoglycolate processing in human cells with a hereditary mutation in tyrosyl-DNA phosphodiesterase (TDP1)"</p><p>Nucleic Acids Research 2005;33(1):289-297.</p><p>Published online 12 Jan 2005</p><p>PMCID:PMC546157.</p><p>© 2005, the authors © </p> The substrate was treated with 0.3, 1 or 3 mg/ml of each whole-cell extract (), or with 0.14, 1.4 or 5 mg/ml of each nuclear extract () for 2 h in the presence of EDTA. Following heat denaturation of cellular proteins, the substrate was treated with PNKP to convert 3′-phosphate to 3′-OH termini. Titration with various concentrations of extract () showed that whole-cell extracts (closed circles) contained about twice as much PG-processing activity as nuclear extracts (open squares)