HMGB1/2 can target DNA for illegitimate cleavage by the RAG1/2 complex

Ming Zhang, Patrick Swanson

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Background: 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. Results: 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. Conclusion: 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.

Original languageEnglish
Article number24
JournalBMC Molecular Biology
Volume10
DOIs
StatePublished - Mar 24 2009

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HMGB2 Protein
HMGB1 Protein
DNA Cleavage
Genetic Recombination
Protein Sorting Signals
Chromosome Pairing
Inverted Repeat Sequences
V(D)J Recombination
Antigen Receptors
Base Pairing
HMG-Box Domains
DNA
High Mobility Group Proteins
Genetic Translocation
Proteins
Double-Stranded DNA Breaks
Proto-Oncogenes
Conserved Sequence
DNA-Binding Proteins
Plasmids

All Science Journal Classification (ASJC) codes

  • Molecular Biology

Cite this

HMGB1/2 can target DNA for illegitimate cleavage by the RAG1/2 complex. / Zhang, Ming; Swanson, Patrick.

In: BMC Molecular Biology, Vol. 10, 24, 24.03.2009.

Research output: Contribution to journalArticle

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abstract = "Background: 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. Results: 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. Conclusion: 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.",
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