Both High Mobility Group (HMG)-boxes and the Acidic Tail of HMGB1 Regulate Recombination-activating Gene (RAG)-mediated Recombination Signal Synapsis and Cleavage in Vitro

Tina Madathiparambil,Serge Bergeron,Patrick C. Swanson

doi:10.1074/jbc.m503063200

Tina Madathiparambil, Serge Bergeron + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m503063200

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Abstract

RAG-1 and RAG-2 initiate V(D)J recombination through synapsis and cleavage of a 12/23 pair of V(D)J recombination signal sequences (RSS). RAG-RSS complex assembly and activity in vitro is promoted by high mobility group proteins of the "HMG-box" family, exemplified by HMGB1. How HMGB1 stimulates the DNA binding and cleavage activity of the RAG complex remains unclear. HMGB1 contains two homologous HMG-box DNA binding domains, termed A and B, linked by a stretch of basic residues to a highly acidic C-terminal tail. To identify determinants of HMGB1 required for stimulation of RAG-mediated RSS binding and cleavage, we prepared an extensive panel of mutant HMGB1 proteins and tested their ability to augment RAG-mediated RSS binding and cleavage activity. Using a combination of mobility shift and in-gel cleavage assays, we find that HMGB1 promotes RAG-mediated cleavage largely through the activity of box B, but optimal stimulation requires a functional A box tethered in the correct orientation. Box A or B mutants fail to promote RAG synaptic complex formation, but this defect is alleviated when the acidic tail is removed from these mutants.

Highlights

During lymphocyte development, antigen receptor genes undergo a series of DNA rearrangements to generate functional exons encoding the antigen binding domains of these receptors [1]
HMGB1 was found to be required for recombination-activating gene (RAG)-mediated synapsis and cleavage of intact recombination signal sequences (RSS) pairs according to the 12/23 rule, and 12/23-regulated cleavage of nicked RSS pairs, despite not being rigorously required for synapsis of nicked substrates [18]
Structural, and biochemical characterization of HMGB1 and related high mobility group (HMG)-box proteins suggest that HMG box A, box B, the basic linker, and the acidic tail of HMGB1 are encompassed within the amino acid residues shown in Fig. 1A [10]

Summary

Introduction

Antigen receptor genes undergo a series of DNA rearrangements to generate functional exons encoding the antigen binding domains of these receptors [1]. Despite being incorporated into RAG-RSS complexes, individual HMGbox domain proteins and full-length HMGB1 bearing mutations in box B fail to stimulate RAG-mediated DNA cleavage of single RSS substrates.

Results

Conclusion