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. 2012 Aug 13;40(20):10394–10407. doi: 10.1093/nar/gks763

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© The Author(s) 2012. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 6. — Analysis of RAD6A∼Ub_n formation. (A) Formation of thiol-linked Ub chains on RAD6A using the minimal set of factors. The reactions were reconstituted with mpssDNA, E1, RAD6A–RAD18, MMS2–UBC13, HLTF and Ub. Reaction products were treated with or without a reducing agent and then analyzed by immunoblot using an anti-RAD6 antibody. –, omitted factor. (B) Evidence that the multiple thiol-linked bands on RAD6A are Lys63 linked Ub chains. Reaction products untreated by a reducing agent were analyzed by immunoblot using an anti-RAD6 antibody. The positions of RAD6A∼Ub_n and RAD6A∼^FLAGUb_n are indicated by dots and stars, respectively. (C) Effect of mutants on thiol-linked Ub chain formation on RAD6A. Reaction products untreated by a reducing agent were analyzed by immunoblot using an anti-RAD6 antibody. –, omitted factor; CA for ^HisHLTF, ^HisHLTF^C760A; IRAA, ^HisRAD18^I50A/R51A; CA for UBC13, UBC13^C87A; CA for RAD6A and RAD6A^C88A. (D) Proposed mechanism of RAD6A∼Ub_n formation. A red arrow depicts the direction of Ub_n movement. MMS2 and RAD18 were included in the reactions but omitted from the schematic. (E) Purified RAD6A∼^HisUb–^HisRAD18 and RAD6A∼^HisUb^K63R–^HisRAD18 complexes treated with or without a reducing agent were analyzed by SDS–PAGE followed by CBB staining. (F and G) PCNA monoubiquitination reactions were reconstituted with PCNA, mpssDNA, RFC and the indicated complexes (K, RAD6A∼^HisUb–^HisRAD18; or R, RAD6A∼^HisUb^K63R–^HisRAD18) for the indicated times. Reaction products treated with (F) or without (G) a reducing agent were analyzed by immunoblot using an anti-PCNA antibody (F) or an anti-RAD6 antibody (G). (H) Analysis of the Ub transfer reaction from UBC13∼Ub to RAD6∼Ub. Reactions were performed with mpssDNA, E1, MMS2–UBC13, HLTF, Ub^K63R, and either RAD6–^HisRAD18, RAD6A∼^HisUb–^HisRAD18, or RAD6A∼^HisUb^K63R–^HisRAD18 for 2 min. RAD6 molecules shown by (+) were carried from partial charging reactions, as shown in (E). Reaction products untreated with a reducing agent were analyzed by immunoblot using an anti-RAD6 antibody. Lane 10, standard reaction products. (I) Analysis of the Ub chain transfer reaction from RAD6∼Ub₂ to PCNA. Reactions were performed with PCNA, mpssDNA, RFC, E1, MMS2–UBC13, HLTF, Ub^K63R, and either RAD6–^HisRAD18, RAD6∼^HisUb–^HisRAD18, or RAD6∼^HisUb^K63R–^HisRAD18 for 2 min and then analyzed by immunoblot using an anti-PCNA antibody. Lane 10, standard reaction products.