Cooperative recruitment of HMGB1 during V(D)J recombination through interactions with RAG1 and DNA

Abstract

During V(D)J recombination, recombination activating gene (RAG)1 and RAG2 bind and cleave recombination signal sequences (RSSs), aided by the ubiquitous DNA-binding/-bending proteins high-mobility group box protein (HMGB)1 or HMGB2. HMGB1/2 play a critical, although poorly understood, role in vitro in the assembly of functional RAG–RSS complexes, into which HMGB1/2 stably incorporate. The mechanism of HMGB1/2 recruitment is unknown, although an interaction with RAG1 has been suggested. Here, we report data demonstrating only a weak HMGB1–RAG1 interaction in the absence of DNA in several assays, including fluorescence anisotropy experiments using a novel Alexa488-labeled HMGB1 protein. Addition of DNA to RAG1 and HMGB1 in fluorescence anisotropy experiments, however, results in a substantial increase in complex formation, indicating a synergistic binding effect. Pulldown experiments confirmed these results, as HMGB1 was recruited to a RAG1–DNA complex in a RAG1 concentration-dependent manner and, interestingly, without strict RSS sequence specificity. Our finding that HMGB1 binds more tightly to a RAG1–DNA complex over RAG1 or DNA alone provides an explanation for the stable integration of this typically transient architectural protein in the V(D)J recombinase complex throughout recombination. These findings also have implications for the order of events during RAG–DNA complex assembly and for the stabilization of sequence-specific and non-specific RAG1–DNA interactions.

HMGB1 in health and disease

“…Following DNA cleavage by RAG, the nicked strand is converted to a hairpin and then the broken ends are joined by several proteins involved in DNA damage responses (Gellert, 2002). HMGB1 plays an essential role in V(D)J recombination by formation of RAG-RSS-HMG complexes to enhance RAG1/RAG2 activity (Agrawal and Schatz, 1997; Dai et al, 2005; Grundy et al, 2009; Little et al, 2013; Swanson, 2002a, b). The knowledge of their interconnection is essential for our understanding of immune cell development.…”

High mobility group (HMG) proteins: Modulators of chromatin structure and DNA repair in mammalian cells

“…Swanson [162] further demonstrated that recombinant mammalian HMGB1 binds to the 12/23 substrates in vitro, and that it does so in a complex with the RAG1 and 2 proteins, again mimicking the early steps in the recombination process. More recent experiments have suggested that HMGB1 is initially recruited to a complex of RAG proteins plus DNA to form a tertiary complex that is stable throughout the recombination process [213]. Aidinis et al [214] were among the first to propose that RAG proteins recruit HMG1 and HMG2 to facilitate recombination signal sequence binding and to enhance the intrinsic DNA-bending activity of RAG1 and RAG2 proteins.…”

Reduction in replication‐independent endogenous DNA double‐strand breaks promotes genomic instability during chronological aging in yeast

“…Interestingly, HMGB1 can both maintain and produce EDSBs. HMGB1 is a known protein that plays a role in maintaining EDSBs generated by the V(D)J recombination process (22)(23)(24) and possesses deoxyribose phosphate lyase activity (25). RIND-EDSBs are located in specific genomic regions, that is, their occurrences are nonrandom.…”