Table I.

Summary of activity of HMGB1 proteins in RAG binding and cleavage assays

HMGB1 protein	Single RSS bindinga	Supershift of SC1 and/or SC2b	RSS cleavage (MT SC1/WT SC1)c
			Nick	Hairpin	Ab nick
WT	+; 150 ng	+(SC1/SC2)	1	1	1
A	+ + +; 50 ng	+(SC1/SC2)	0.15	0.10	1.64
B′	–; 150 ng	– (up to 640 ng)	0.07	0.09	1.62
B	ND	+(SC1/SC2)
mtA	+; 150 ng	+(SC1/SC2)	0.50	0.42	2.56
mtB	+; 150 ng	+(SC1/SC2)	0.17	0.09	2.56
mtAB	–/+; 150 ng	–/+ (SC1), + (SC2)	0.12	0.09	2.79
Basic	+ + +; 50 ng	+(SC1/SC2)	0.30	0.73	0.48
Tailless	+ + +; 50 ng	+(SC1/SC2)	0.62	0.93	0.43
Tailless mtA	+ +; 50 ng	+(SC1/SC2)	0.48	0.53	0.76
Tailless mtB	+ +; 50 ng	+(SC1/SC2)	0.16	0.09	1.19
Shuffled	+ + +; 50 ng	+(SC1/SC2)	0.07	0.11	1.57

HMGB1 protein	PC formationd	RSS cleavage (MT PC/WT PC)e
		Nick	Hairpin	Ab nick
WT	+ +	1	1	1
A	+ (weak)	ND	ND	ND
B′	–	ND	ND	ND
B	+ (weak)	ND	ND	ND
mtA	–	0.60	0.23	2.49
mtB	–	0.30	0.07	4.55
mtAB	–	ND	ND	ND
Basic	+ +	0.64	1.26	0.91
Tailless	+ +	0.7	1.15	0.96
Tailless mtA	+ +	0.41	0.35	1.20
Tailless mtB	+	ND	ND	ND
Shuffled	+	0.47	0.71	1.26

^aBinding to 23-RSS in the absence of RAGs at indicated amounts of HMGB1 protein using EMSA (Fig. 3); –, no detectable binding; –/+, weak binding; +, binding evident, but little oligomerization; + +, moderate oligomerization; + + +, extensive oligomerization. ND, not done.

^bAbility to supershift SC1 and/or SC2 at amounts of HMGB1 protein used to detect single RSS binding (Fig. 3); –, no supershift detected (up to indicated amount of HMGB1 protein); –/+, impaired supershift; +, supershift comparable to WT.

^cAbundance of cleavage products isolated from SC1 complex using in-gel cleavage assay relative to WT HMGB1 (Fig. 4).

^dAbility to support PC formation detectable by EMSA (Fig. 5). –, no PC formation detected; +, PC formation detectable, but bands diffuse or weak; + +, PC clearly evident.

^eAbundance of cleavage products isolated from PC using in-gel cleavage assay relative to WT HMGB1 (Fig. 6). ND, not done.