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Figure 3 | BMC Molecular Biology

Figure 3

From: HMGB1/2 can target DNA for illegitimate cleavage by the RAG1/2 complex

Figure 3

Oligonucleotide substrates containing bps6197 are bound and cleaved by the RAG proteins in vitro. (A) Radiolabeled 23-RSS or bps6197 oligonucleotide substrates were incubated in an in vitro cleavage reaction containing WT cMR1/cMR2 with or without added HMGB1 and/or cold partner 12- or 23-RSS (0.1 or 1.0 pmol) as indicated. Reaction products were fractionated on a sequencing gel in parallel with radiolabeled markers corresponding to predicted nick and hairpin products. The percentage of appropriately sited nick (%N) and hairpin (%HP) products as well as aberrant nicks (%Abnicks) are quantified below the gel and are representative of independent experiments. (B) (Left panel) Radiolabeled 23-RSS or bps6197 oligonucleotide substrates were incubated with WT cMR1/cMR2 in binding reactions in the absence or presence of HMGB1 and increasing amounts (0.1, 1.0, or 10 pmol) of cold 23-RSS as a competitor. (Right panel) WT cMR1/cMR2 was incubated with a radiolabeled 23-RSS substrate with or without HMGB1 and increasing amounts (0.1, 1.0, or 1.0 pmol) of cold 23-RSS, bps6197, Hox11 or non-specific (NS) oligonucleotide substrates as a competitor. Protein-DNA complexes were separated by EMSA.

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