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. Author manuscript; available in PMC: 2010 Sep 1.
Published in final edited form as: Biochem J. 2009 Aug 13;422(2):329–341. doi: 10.1042/BJ20090534

Figure 4. Argonaute2 is a RNA-dependent endonuclease that requires magnesium and the integrity of the DDH catalytic domain.

Figure 4

(A) Let-7a-directed cleavage of mRNA by Argonaute2 requires magnesium. Reactions (20 µl) containing 50 mM Tris, pH 7.5, 50 mM KCl, 2 nM let-7a RNA and 200 nM GST–Argonaute2 were preincubated for 30 min in the presence of either EDTA or MgCl2 (as indicated). 5′-end-labelled target mRNA (1 nM) was added to each reaction followed by a 15 min incubation. Formamide loading buffer was added and reactions were analysed by 12% polyacrylamide run under denaturing conditions. M, marker nucleotide ladder. (B) The DDH domain of Argonaute2 is essential. Reactions containing 2 nM let-7a and 75, 150 or 375 nM GST–Argonaute2 or active site mutants D597A, D699A and H807A (as indicated) were carried out as described above. (C) Argonaute2 is a RNA-dependent endonuclease. Reactions containing 200 nM GST–Argonaute2 and let-7a ribose or let-7a deoxyribose (as indicated) were carried out as described above.