HMG-box domain stimulation of RAG1/2 cleavage activity is metal ion dependent

Abstract

Background: RAG1 and RAG2 initiate V(D)J recombination by assembling a synaptic complex with a pair of antigen receptor gene segments through interactions with their flanking recombination signal sequence (RSS), and then introducing a DNA double-strand break at each RSS, separating it from the adjacent coding segment. While the RAG proteins are sufficient to mediate RSS binding and cleavage in vitro, these activities are stimulated by the architectural DNA binding and bending factors HMGB1 and HMGB2. Two previous studies (Bergeron et al., 2005, andDai et al., 2005) came to different conclusions regarding whether only one of the two DNA binding domains of HMGB1 is sufficient to stimulate RAG-mediated binding and cleavage of naked DNA in vitro. Here we test whether this apparent discrepancy is attributed to the choice of divalent metal ion and the concentration of HMGB1 used in the cleavage reaction.

Differential Reaction Kinetics, Cleavage Complex Formation, and Nonamer Binding Domain Dependence Dictate the Structure-Specific and Sequence-Specific Nuclease Activity of RAGs

“…,37 A single 23-RSS cleavage is enhanced by HMGB1 in the presence of Mn 2+ , but not Mg 2+ , whereas coupled cleavage and binding on 23-RSS were enhanced by HMGB1 in Mg2+ . In the present study, we found that HMGB1 does not affect the cleavage of bubble substrates in the presence of either Mg 2+ or Mn 2+(Fig.…”

V(D)J Recombination: Mechanisms of Initiation

“…Based on their known DNA binding and bending properties (137), boxes A and B are speculated to occupy sites in or near the heptamer and nonamer, respectively. The tandem arrangement of the HMGB domains may provide bridging needed between the two sites in the SC to stimulate binding, but once synapsis with a partner RSS has been achieved, a conformational change may occur to stabilize the complex and obviate the function of one of the two HMGB domains (79). The HMGB1 stoichiometry in precleavage RAG-RSS complexes remains to be formally defined, but recent biochemical characterization of the SEC suggests the ratio of RAG1:RAG2:HMGB1 is approximately 2:2:1 in this complex (54).…”

Structural Analysis of HMGD–DNA Complexes Reveals Influence of Intercalation on Sequence Selectivity and DNA Bending

“…They participate in the function of chromatin remodeling by interacting with nucleosomes 5; 6; 7; 8;9, altering the interaction of nucleosomes with DNA 10; 11;12, and aiding chromatin remodeling machinery 10; 13; 14. HMGB1/2 facilitates V(D)J recombination by interacting with recombination activating genes (RAG) proteins and enhancing DNA cleavage rates 15; 16; 17; 18. The HMGB proteins regulate transcription through interactions with many types of transcription factors 19; 20; 21;22; 23; 24 and proteins necessary for efficient transcript elongation 25.…”