RAG and HMGB1 Proteins: Purification and Biochemical Analysis of Recombination Signal Complexes

Bergeron, Serge; Anderson, Dirk K.; Swanson, Patrick C.

doi:10.1016/s0076-6879(06)08032-3

Cited by 36 publications

(78 citation statements)

References 46 publications

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“…Sequences for the substrates used in this study are shown in Table 1 (top strand only). To prepare intact or nicked duplex substrates, the top or bottom strand oligonucleotides were radiolabeled with [␥-32 P]ATP using T4 polynucleotide kinase, annealed to their unlabeled complementary strand(s), and the duplex was purified on a native polyacrylamide gel as described previously (25). Plasmid V(D)J recombination substrates containing either consensus or cryptic recombination signals were generously provided by Michael Lieber and are indicated in Table 1 (14).…”

Section: Methodsmentioning

confidence: 99%

“…Protein Expression and Purification-Wild-type, catalytically inactive (D600A) or hyperactive (E649A) forms of core (residues 384 -1040) RAG1 and core (residues 1-387) RAG2 were coexpressed in 293 cells as maltose-binding protein fusion proteins (cMR1 and cMR2, respectively) and purified as described previously (25). The yields of wild-type and mutant RAG proteins were all similar.…”

Section: Methodsmentioning

confidence: 99%

“…In some experiments, preparations of core RAG1 and full-length RAG2 or full-length RAG1 and core RAG2 were used (cMR1/FLMR2 or FLMR1/cMR2, respectively) (26). Full-length HMGB1 (high mobility group box 1) was expressed in Escherichia coli strain BL21(DE3)pLysS and purified using a combination of immobilized metal affinity chromatography and ion exchange chromatography as described previously (25).…”

Section: Methodsmentioning

confidence: 99%

“…Oligonucleotide Cleavage and Binding Assays-RAG-mediated cleavage of oligonucleotide substrates was analyzed using an in vitro cleavage assay performed under permissive conditions containing Me 2 SO to promote RSS cleavage as described (25). Briefly, coexpressed wild-type or mutant RAG1/RAG2 proteins (100 ng) and HMGB1 (300 ng, where indicated) were incubated with substrate DNA (ϳ0.02 pmol) in a 10-l reaction containing sample buffer (25 mM MOPS-KOH (pH 7.0), 60 mM potassium glutamate, 100 g/ml bovine serum albumin, 20% Me 2 SO, and 1 mM MgCl 2 ).…”

Section: Methodsmentioning

confidence: 99%

“…Samples were incubated at 37°C for 1 h, and the reaction products were visualized and quantified from dried sequencing gels using a Molecular Dynamics Storm 860 PhosphorImager running the ImageQuant software. Direct RAG binding to cRSS substrates was analyzed by electrophoretic mobility shift assay (EMSA) using the same buffer conditions as those used in the in vitro cleavage assay, except that CaCl 2 replaced MgCl 2 in the binding reaction to avoid substrate cleavage (25). For competition experiments shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

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V(D)J Recombinase Binding and Cleavage of Cryptic Recombination Signal Sequences Identified from Lymphoid Malignancies

Zhang

Swanson

2008

Journal of Biological Chemistry

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To understand the molecular basis for these large differences, we investigated the binding and cleavage of these cRSSs by the RAG1/2 proteins that initiate V(D)J recombination. We find that the RAG proteins comparably bind all cRSSs tested, albeit more poorly than a consensus RSS. We show that four cRSSs that support levels of V(D)J recombination above background levels in cell culture (LMO2, TAL1, Ttg-1, and SIL) are also cleaved by the RAG proteins in vitro with efficiencies ranging from 18 to 70% of a consensus RSS. Cleavage of LMO2 and Ttg-1 by the RAG proteins can also be detected in cell culture using ligation-mediated PCR. In contrast, Hox11 and SCL are nicked but not cleaved efficiently in vitro, and cleavage at other adventitious sites in plasmid substrates may also limit the ability to detect recombination activity at these cRSSs in cell culture.The antigen binding domains of immunoglobulins and T cell receptors are encoded in germ line arrays of V, D, and J gene segments that are assembled into functional variable exons by V(D)J recombination during lymphocyte development (1). The site of recombination is directed by a recombination signal sequence (RSS) 2 that flanks each receptor gene segment and consists of a conserved heptamer (consensus 5Ј-CACAGTG-3Ј) and nonamer (consensus 5Ј-ACAAAAACC-3Ј) separated by either 12 or 23 Ϯ 1 nucleotides of more highly varied sequence. V(D)J recombination can be conceptually divided into two phases, a cleavage phase and a joining phase (2). In the cleavage phase, the lymphoid cell-specific RAG1 and RAG2 (recombination activating gene-1 and -2, respectively) proteins assemble a multiprotein synaptic complex with two RSSs (generally one 12-RSS and one 23-RSS) and introduce a DNA double strand break at each RSS between the heptamer and adjacent coding segment via a nick-hairpin mechanism to yield a blunt 5Ј-phosphorylated signal end and a coding end terminating in a covalently sealed DNA hairpin structure. In the joining phase, the signal ends are generally joined precisely to form signal joints, and the coding ends are processed and joined to form coding joints that typically contain nucleotide additions or deletions at the junction. These processes are normally mediated by components of the nonhomologous end-joining DNA repair pathway.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

V(D)J Recombinase Binding and Cleavage of Cryptic Recombination Signal Sequences Identified from Lymphoid Malignancies

Zhang

Swanson

2008

Journal of Biological Chemistry

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Early Steps of V(D)J Rearrangement: Insights from Biochemical Studies of RAG-RSS Complexes

Swanson

Kumar

Raval

2009

Advances in Experimental Medicine and Biology

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High mobility group proteins and their post-translational modifications

Zhang

Wang

2008

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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The high mobility group (HMG) proteins, including HMGA, HMGB and HMGN, are abundant and ubiquitous nuclear proteins that bind to DNA, nucleosome and other multi-protein complexes in a dynamic and reversible fashion to regulate DNA processing in the context of chromatin. All HMG proteins, like histone proteins, are subjected to extensive post-translational modifications (PTMs), such as lysine acetylation, arginine/lysine methylation and serine/threonine phosphorylation, to modulate their interactions with DNA and other proteins. There is a growing appreciation for the complex relationship between the PTMs of HMG proteins and their diverse biological activities. Here, we reviewed the identified covalent modifications of HMG proteins, and highlighted how these PTMs affect the functions of HMG proteins in a variety of cellular processes.

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RAG and HMGB1 Proteins: Purification and Biochemical Analysis of Recombination Signal Complexes

Cited by 36 publications

References 46 publications

V(D)J Recombinase Binding and Cleavage of Cryptic Recombination Signal Sequences Identified from Lymphoid Malignancies

V(D)J Recombinase Binding and Cleavage of Cryptic Recombination Signal Sequences Identified from Lymphoid Malignancies

Early Steps of V(D)J Rearrangement: Insights from Biochemical Studies of RAG-RSS Complexes

High mobility group proteins and their post-translational modifications

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