Figure 2: WRN c. 2500C > T (p. R834C) selectively reduces DNA helicase, but not DNA exonuclease, activity. | Scientific Reports

Figure 2: WRN c. 2500C > T (p. R834C) selectively reduces DNA helicase, but not DNA exonuclease, activity.

From: Homozygosity for the WRN Helicase-Inactivating Variant, R834C, does not confer a Werner syndrome clinical phenotype

Figure 2

Endogenous WRN in lysates (1 mg total protein) prepared from control (CC), heterozygous (CT), and homozygous (TT) cells from four pedigrees (F14, F19, F26, and F33) was affinity purified with a polyclonal anti-WRN antibody. Immunoprecipitates containing WRN were assayed for either DNA helicase (a) or DNA exonuclease (b) activity. Unwinding activity was measured using a 3′-end blocked 20/46 nt partial DNA duplex substrate, while exonuclease activity was assessed in the presence of ATPγS using the unblocked substrate; both substrates were 5′-end labeled using γ[32P]ATP and T4 polynucleotide kinase. Reactions were incubated at 37 °C for 20 min and aliquots were electrophoresed through native 12% (helicase) or denaturing 14% (exonuclease) polyacrylamide gels. Radioactivity in bands was visualized on a PhosphorImager, and band intensities were quantified using Image J software. Enzymatic activity in heterozygous and homozygous cells was normalized to that of within-pedigree controls.

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