Nucleic Acids Research, 2000, Vol. 28, No. 5 1228-1236
© 2000 Oxford University Press
A highly ordered structure in V(D)J recombination cleavage complexes is facilitated by HMG1
Medical College of Georgia, Institute of Molecular Medicine and Genetics, CB-2803, Augusta, GA 30912, USA
Central to understanding the process of V(D)J recombination is appreciation of the protein–DNA complex which assembles on the recombination signal sequences (RSS). In addition to RAG1 and RAG2, the protein HMG1 is known to stimulate the efficiency of the cleavage reaction. Using electrophoretic mobility shift analysis we show that HMG1 stimulates the in vitro assembly of a stable complex with the RAG proteins on each RSS. We use UV crosslinking studies of this complex with azido-phenacyl derivatized probes to map the contact sites between the RAG proteins, HMG1 derivatives and the RSS. We find that the RAG proteins make contacts at the nonamer, heptamer and adjacent coding region. The HMG1 protein by itself appears to localize at the 3' side of the nonamer, but a cooperative complex with the RAG proteins is positioned at the 3' side of the heptamer and adjacent spacer in the 12RSS. In the complex with RAG proteins, HMG1 is positioned primarily in the spacer of the 23RSS. We suggest that bends introduced into these DNA substrates at specific locations by the RAG proteins and HMG1 may help distinguish the 12RSS from the 23RSS and may therefore play an important role in the coordinated reaction.
* To whom correspondence should be addressed. Tel: +1 706 721 8761; Fax: +1 706 721 8752; Email: moshe@immag.mcg.edu
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