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

Department of Medical Microbiology and Immunology, Creighton University Medical Center, 2500 California Plaza, Omaha, NE, USA.
BMC Molecular Biology (Impact Factor: 2.19). 02/2008; 9(1):32. DOI: 10.1186/1471-2199-9-32
Source: PubMed


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, and Dai 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.
We show here that single HMG-box domains of HMGB1 stimulate RAG-mediated RSS cleavage in a concentration-dependent manner in the presence of Mn2+, but not Mg2+. Interestingly, the inability of a single HMG-box domain to stimulate RAG-mediated RSS cleavage in Mg2+ is overcome by the addition of partner RSS to promote synapsis. Furthermore, we show that mutant forms of HMGB1 which otherwise fail to stimulate RAG-mediated RSS cleavage in Mg2+ can be substantially rescued when Mg2+ is replaced with Mn2+.
The conflicting data published previously in two different laboratories can be substantially explained by the choice of divalent metal ion and abundance of HMGB1 in the cleavage reaction. The observation that single HMG-box domains can promote RAG-mediated 23-RSS cleavage in Mg2+ in the presence, but not absence, of partner RSS suggests that synaptic complex assembly in vitro is associated with conformational changes that alter how the RAG and/or HMGB1 proteins bind and bend DNA in a manner that functionally replaces the role of one of the HMG-box domains in RAG-HMGB1 complexes assembled on a single RSS.

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    • "Interestingly, except for Box B', these proteins remain capable of stimulating 12-RSS cleavage in these substrates relative to samples containing cMR1/cMR2 alone. This outcome is consistent with our recent data showing that despite failing to stimulate RAG-mediated cleavage on an isolated 23-RSS oligonucleotide substrate in Mg2+, a single HMG-box domain can promote 23-RSS cleavage when a partner 12-RSS is present [27]. In this study, either the 23-RSS or bps6197 may serve in cis as the partner to the 12-RSS in the long DNA substrate. "
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    ABSTRACT: V(D)J recombination is initiated in antigen receptor loci by the pairwise cleavage of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins via a nick-hairpin mechanism. The RSS contains highly conserved heptamer (consensus: 5'-CACAGTG) and nonamer (consensus: 5'-ACAAAAACC) motifs separated by either 12- or 23-base pairs of poorly conserved sequence. The high mobility group proteins HMGB1 and HMGB2 (HMGB1/2) are highly abundant architectural DNA binding proteins known to promote RAG-mediated synapsis and cleavage of consensus recombination signals in vitro by facilitating RSS binding and bending by the RAG1/2 complex. HMGB1/2 are known to recognize distorted DNA structures such as four-way junctions, and damaged or modified DNA. Whether HMGB1/2 can promote RAG-mediated DNA cleavage at sites lacking a canonical RSS by targeting or stabilizing structural distortions is unclear, but is important for understanding the etiology of chromosomal translocations involving antigen receptor genes and proto-oncogene sequences that do not contain an obvious RSS-like element. Here we identify a novel DNA breakpoint site in the plasmid V(D)J recombination substrate pGG49 (bps6197) that is cleaved by the RAG proteins via a nick-hairpin mechanism. The bps6197 sequence lacks a recognizable heptamer at the breakpoint (5'-CCTGACG-3') but contains a nonamer-like element (5'-ACATTAACC-3') 30 base pairs from the cleavage site. We find that RAG-mediated bps6197 cleavage is promoted by HMGB1/2, requiring both HMG-box domains to be intact to facilitate RAG-mediated cleavage, and is stimulated by synapsis with a 12-RSS. A dyad-symmetric inverted repeat sequence lying 5' to the breakpoint is implicated as a target for HMGB1/2 activity. We have identified a novel DNA sequence, called bps6197, that supports standard V(D)J-type cleavage despite the absence of an apparent heptamer motif. Efficient RAG-mediated bps6197 cleavage requires the presence of HMGB1/2, is stimulated by synapsis with a 12-RSS partner, and is directed in part by an inverted repeat sequence adjacent to the DNA cleavage site. These results have important implications for understanding how the RAG proteins can introduce a DNA double-strand break at DNA sequences that do not contain an obvious heptamer-like motif.
    Full-text · Article · Feb 2009 · BMC Molecular Biology
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    ABSTRACT: V(D)J recombination is initiated by the synapsis and cleavage of a complementary (12/23) pair of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins. Our understanding of these processes has been greatly aided by the development of in vitro biochemical assays of RAG binding and cleavage activity. Accumulating evidence suggests that synaptic complex assembly occurs in a step-wise manner and that the RAG proteins catalyze RSS cleavage by mechanisms similar to those used by bacterial transposases. In this chapter we will review the molecular mechanisms of RAG synaptic complex assembly and 12/23-regulated RSS cleavage, focusing on recent advances that shed new light on these processes.
    No preview · Article · Feb 2009 · Advances in Experimental Medicine and Biology
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    ABSTRACT: HMGB proteins are members of the High Mobility Group (HMG) superfamily, possessing a unique DNA-binding domain, the HMG-box, which can bind non-B-type DNA structures (bent, kinked and unwound) with high affinity, and also distort DNA by bending/looping and unwinding. HMGBs (there are four HMGBs in mammals, HMGB1-4) are highly abundant and ubiquitously expressed non-histone proteins, acting as DNA chaperones influencing multiple processes in chromatin such as transcription, replication, recombination, DNA repair and genomic stability. Although HMGB1 is a nuclear protein, it can be secreted into the extracellular milieu as a signaling molecule when cells are under stress, in particular, when necrosis occurs. Mammalian HMGBs contain two HMG-boxes arranged in tandem, share more than 80% identity and differ in the length (HMGB1-3) or absence (HMGB4) of the acidic C-tails. The acidic tails consist of consecutive runs of only Glu/Asp residues of various length, and modulate the DNA-binding properties and functioning of HMGBs. HMGBs are subject to post-translational modifications which can fine-tune interactions of the proteins with DNA/chromatin and determine their relocation from the nucleus to the cytoplasm and secretion. Association of HMGBs with chromatin is highly dynamic, and the proteins affect the chromatin fiber as architectural factors by transient interactions with nucleosomes, displacement of histone H1, and facilitation of nucleosome remodeling and accessibility of the nucleosomal DNA to transcription factors or other sequence-specific proteins.
    Full-text · Article · Jan 2010 · Biochimica et Biophysica Acta
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