Amino acid substitutions progressively restore HMGB1 binding and RAG1 activity. (A) HMGB1 binds less well to R401W/12-RSS complexes. WT or R401W mutant RAG1 complexes were formed with a labeled 12-RSS in the presence or absence of His-tagged HMGB1. A 23-RSS oligonucleotide partner was present in all cases. Supershifted complexes formed on addition of an anti-His tag antibody are indicated. The mobilities of SC1, SC2, HSC1, and HSC2 are shown. (B) Increasing amounts of HMGB1 do not restore cutting with R401W. RAG cutting reactions were performed with WT RAG1 or R401W in the presence of increasing amounts of HMGB1 (0, 25, 50, 100, 200, 400, 800 nmoles). A 23-RSS substrate was used, as HMGB1 has the greatest effect at this type of RSS. It is notable that in the absence of HMGB1, cutting by WT RAG1 is not detectable (lane 2). (C) R401W has catalytic activity. RAG cutting reactions were performed using a single labeled 12-RSS in the presence of manganese. (D) HSC1 and HSC2 complexes become increasingly like WT RAG1 complexes with R401L and R401K. RAG binding to a labeled 12- or 23-RSS in the presence of an unlabeled partner RSS and HMGB1, as indicated. The gel was run for longer than Figure 2B to better separate the complexes; a cropped image is shown to highlight the mobility differences. (E) RAG cutting with R401W, R401L, and R401K. RAG cutting was performed with a labeled 12-RSS or 23-RSS, indicated by the asterisk, in the presence of unlabeled partner and the RAG1 proteins indicated, with or without HMGB1. The percentage cutting is given beneath the gel. (F) R401K recombination activity is close to WT RAG1 levels in vivo. Recombination was monitored using pJH299 and equivalent levels of RAG1 proteins (Figure 1B,D). Recombination levels are shown relative to WT RAG1. n = 3; error bars show SEM.