RAG and HMGB1 create a large bend in the 23RSS in the V(D)J recombination synaptic complexes

Ciubotaru, Mihai; Trexler, Adam J.; Spiridon, Laurentiu N.; Surleac, Marius; Rhoades, Elizabeth; Petrescu, Andrei‐Jose; Schatz, David G.

doi:10.1093/nar/gks1294

Cited by 22 publications

(50 citation statements)

References 59 publications

(116 reference statements)

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“…5B and Fig. S4B), which is consistent with previous studies (6,(24)(25)(26). We also find the K d values for the 12RSS and the 23RSS in the presence of RAG1/2c and HMGB1 are within error of K d values previously reported that were derived from the turnover number (k cat ) (27).…”

Section: Resultssupporting

confidence: 92%

“…This observation is consistent with the HMGB1 dependence of the large bends detected in 12RSS-RAG-HMGB1 or 23RSS-RAG-HMGB1 complexes, although those studies used short oligonucleotide substrates (25,26). We also used this reduction in DNA tether length to determine the K d for the 12RSS and the 23RSS in the presence of RAG1/2c and HMGB1 and found that HMGB1 decreased both similar, consistent with previous observations (6,27).…”

Section: Dynamics Of 12/23 Rule-regulated Bead Release As a Function Ofsupporting

confidence: 90%

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Single-molecule analysis of RAG-mediated V(D)J DNA cleavage

Lovely

Brewster

Schatz

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. We have developed singlemolecule assays to examine RSS binding by RAG1/2 and their cofactor high-mobility group-box protein 1 (HMGB1) as they proceed through the steps of this reaction. These assays allowed us to observe in real time the individual molecular events of RAG-mediated cleavage. As a result, we are able to measure the binding statistics (dwell times) and binding energies of the initial RAG binding events and characterize synapse formation at the single-molecule level, yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage on forming the synapse. Interestingly, we find that the synaptic complex has a mean lifetime of roughly 400 s and that its formation is readily reversible, with only ∼40% of observed synapses resulting in cleavage at consensus RSS binding sites. The recombination-activating genes, RAG1 and RAG2, encode proteins that carry out V(D)J recombination by bringing a 12RSS and a 23RSS together into a paired (or synaptic) complex, nicking the RSS sites adjacent to the heptamer, and converting each nick into a double-strand break, leaving a hairpin at the coding end (Fig. 1). The hairpin is created by nucleophilic attack on the opposing strand by the 3′ hydroxyl group at the nick in a transesterification reaction. After RAG1/2 forms hairpins, it recruits the nonhomologous end joining machinery to repair the ends (1, 2). Since their discovery, the full-length RAG1 and RAG2 proteins have proven difficult to isolate and study in vitro (1). However, core domains (referred to as RAG1c and RAG2c) have been identified by removing a large region from the N terminus of RAG1 (which includes an E3 ubiquitin ligase domain) and a large region from the C terminus of RAG2 (which includes a plant homeodomain) (3, 4). These core proteins have been shown to tetramerize to form RAG1/2c, which retains RSS binding, nicking, and hairpin formation activities (5). High-mobility group-box protein 1 (HMGB1) acts as a cofactor and increases RAG1/2c affinity for the 23RSS (6). HMGB1 is required for paired complex formation and efficient conversion of RAG-mediated nicks into hairpins (7-10).Current in vitro assays that capture the paired complex with RAG1/2c generally place a 12RSS and a 23RSS on two different DNA molecules, typically short oligonucleotides. However, in vivo, antigen receptor loci are assembled using RSSs on the same DNA molecule (1, 11). One prior study captured RAG1/2c and HMGB1 bound to DNA with a 12RSS and a 23RSS on the same substrate using standard bulk assays (12). However, many key mechanistic questions remain unresolved concerning how a diverse immune repertoire is generated by the RAG recombinas...

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Section: Resultssupporting

confidence: 92%

Section: Dynamics Of 12/23 Rule-regulated Bead Release As a Function Ofsupporting

confidence: 90%

Single-molecule analysis of RAG-mediated V(D)J DNA cleavage

Lovely

Brewster

Schatz

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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“…We also note that the combined bending induced by the RAG-HMGB1 complex within the RSS region is substantial enough to bring nonamer and heptamer regions within FRET range of each other (Fig. S3D), which is in agreement with previous ensemble measurements (19,20).…”

Section: Rag Complex Induces Conformational Changes In 12rss and 23rsssupporting

confidence: 91%

“…Transitions to high FRET for substrates in the presence of Mg 2+ are consistent with RAG1/2-induced bending of both 12RSS and 23RSS substrates previously reported in ensemble (i.e., bulk solution) studies ( Fig. 1 A and B) (19,20). The observed bending induced by RAG1/2 differed from HMGB1 activity in two distinct ways: specificity and stability.…”

Section: Rag Complex Induces Conformational Changes In 12rss and 23rsssupporting

confidence: 90%

“…The effect of RAG1/2 complex binding to RSS DNA was previously addressed using biochemical and biophysical techniques (19,20,22,26) and more recently visualized within the PC in structural analysis (see SI Discussion) (14). These studies indicated that binding of HMGB1 and RAG1/2 induced bending in both the 12RSS and 23RSS DNA substrates as well as in each substrate within the PC.…”

Section: Discussionmentioning

confidence: 99%

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Real-time analysis of RAG complex activity in V(D)J recombination

Zagelbaum

Shimazaki

Esguerra

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Single-molecule FRET (smFRET) and single-molecule colocalization (smCL) assays have allowed us to observe the recombination-activating gene (RAG) complex reaction mechanism in real time. Our smFRET data have revealed distinct bending modes at recombination signal sequence (RSS)-conserved regions before nicking and synapsis. We show that high mobility group box 1 (HMGB1) acts as a cofactor in stabilizing conformational changes at the 12RSS heptamer and increasing RAG1/2 binding affinity for 23RSS. Using smCL analysis, we have quantitatively measured RAG1/2 dwell time on 12RSS, 23RSS, and non-RSS DNA, confirming a strict RSS molecular specificity that was enhanced in the presence of a partner RSS in solution. Our studies also provide single-molecule determination of rate constants that were previously only possible by indirect methods, allowing us to conclude that RAG binding, bending, and synapsis precede catalysis. Our real-time analysis offers insight into the requirements for RSS-RSS pairing, architecture of the synaptic complex, and dynamics of the paired RSS substrates. We show that the synaptic complex is extremely stable and that heptamer regions of the 12RSS and 23RSS substrates in the synaptic complex are closely associated in a stable conformational state, whereas nonamer regions are perpendicular. Our data provide an enhanced and comprehensive mechanistic description of the structural dynamics and associated enzyme kinetics of variable, diversity, and joining [V(D)J] recombination. The recombination-activating genes 1 and 2 (RAG1/2) are lymphocyte-specific proteins that mediate recognition and splicing of (V), (D), and (J) segments. The RAG complex, which consists of RAG1/2 and the cofactor high mobility group box 1 (HMGB1), creates DNA double-strand breaks at recombination signal sequences (RSSs) that flank (V), (D), and (J) segments. All RSSs have conserved heptamer and nonamer regions separated by either 12 or 23 bp (12RSS or 23RSS) and act as specific targets for RAG1/2-mediated catalytic activity. It is thought that catalytic activity and initiation of the V(D)J pathway consists of three distinct steps. First, the RAG complex binds and nicks a single RSS substrate to create a single RSS complex (SC). Next, two partner RSSs, generally a 12RSS and 23RSS, synapse to form a paired complex (PC). Finally, RAG1/2 cleaves both RSSs through a transesterification reaction, resulting in hair pinned coding ends that are repaired by nonhomologous end joining (5-12). Whether nicking occurs before or after synapsis is unclear, as is the stability of the synaptic complex (PC). Kinetic studies favor synapsis formation before nicking and a very high stability of the synaptic complex, once formed, but these concepts are based on bulk solution (ensemble) studies, and the specific steps of the reaction mechanism are uncertain due to inevitable averaging (13).Recently, crystallography and electron microscopy techniques have been used to resolve the structures of the SC and PC (14,15). Although these studies provide new...

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Single-molecule studies of high-mobility group B architectural DNA bending proteins

et al. 2016

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Protein–DNA interactions can be characterized and quantified using single molecule methods such as optical tweezers, magnetic tweezers, atomic force microscopy, and fluorescence imaging. In this review, we discuss studies that characterize the binding of high-mobility group B (HMGB) architectural proteins to single DNA molecules. We show how these studies are able to extract quantitative information regarding equilibrium binding as well as non-equilibrium binding kinetics. HMGB proteins play critical but poorly understood roles in cellular function. These roles vary from the maintenance of chromatin structure and facilitation of ribosomal RNA transcription (yeast high-mobility group 1 protein) to regulatory and packaging roles (human mitochondrial transcription factor A). We describe how these HMGB proteins bind, bend, bridge, loop and compact DNA to perform these functions. We also describe how single molecule experiments observe multiple rates for dissociation of HMGB proteins from DNA, while only one rate is observed in bulk experiments. The measured single-molecule kinetics reveals a local, microscopic mechanism by which HMGB proteins alter DNA flexibility, along with a second, much slower macroscopic rate that describes the complete dissociation of the protein from DNA.

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RAG and HMGB1 create a large bend in the 23RSS in the V(D)J recombination synaptic complexes

Cited by 22 publications

References 59 publications

Single-molecule analysis of RAG-mediated V(D)J DNA cleavage

Single-molecule analysis of RAG-mediated V(D)J DNA cleavage

Real-time analysis of RAG complex activity in V(D)J recombination

Single-molecule studies of high-mobility group B architectural DNA bending proteins

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