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. 2017 Oct 19;171(3):588–600.e24. doi: 10.1016/j.cell.2017.09.008

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© 2017 The Author(s)

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Figure S6 — DNA Entrapment by the Kleisin Loop, Related to Figure 5

(A) Residue pairs in Sc Brn1 (green) latch and buckle segments in positions that should be crosslinkable (left panel, S384-S524, M391-T506, D395-S505) or in positions that should not be crosslinkable (right panel, S384-R508, N397-S524, K465-S524) when mutated to cysteine. Corresponding sequence homology pairs in Ct Brn1 (left panel, E514-R629, L521-S611, D525-S610; right panel, E514-Q613, A527-R629, S568-R629).

(B) Analysis of copurified Ct Ycg1_24-1006– His₆-TEV-Brn1_515-634 subcomplexes that either contain no additional cysteine residues (no cys) or additional cysteine pairs engineered into Ct Brn1 as in (listed in (A)) and a target sites for the 3C protease following Brn1 residue P549 (cleavable). Protein complexes were incubated with a 22-bp dsDNA before addition of DMSO solvent or bBBr crosslinker, followed by incubation with 3C protease (+3C) or buffer only (–3C), SDS-PAGE and Coomassie staining of two separate gels in parallel (left panel). EMSA of a 2.1-kb circular DNA with the same Ct Ycg1_24-1006– His₆-TEV-Brn1_515-634 complexes detected by EtBr staining after incubation with DMSO or bBBr crosslinker and protein denaturation (right panel).

(C) Analysis of copurified Ct Ycg1_24-1006– His₆-TEV-Brn1_515-634 complexes without (no cys) or with an additional cysteine pair (Brn1_{E514C, R629C}) engineered into the latch and buckle segments of Brn1 with (cleavable) or without a 3C protease site following Brn1 residue P549 as in (B), using bBBr, BMOE or DTME crosslinkers, followed by incubation with 3C protease (+3C), dithiothreitol (+DTT) or buffer only (see Figures 5B and 5C).

(D) Analysis of copurified Ct Ycs4_3-1222–Ycg1_24-1006– His₆-TEV-Brn1_225-634 complexes without (no cys) or with an additional cysteine pair (Brn1_{E514C, R629C}) engineered into the latch and buckle segments of Brn1 with (cleavable) or without a 3C protease site following Brn1 residue P549 as in (B), using BMOE crosslinker followed by incubation with 3C protease (+3C) or buffer only.