Fluorescence Resonance Energy Transfer Analysis of Recombination Signal Sequence Configuration in the RAG1/2 Synaptic Complex

Ciubotaru, Mihai; Kriatchko, Aleksei N.; Swanson, Patrick C.; Bright, Frank V.; Schatz, David G.

doi:10.1128/mcb.00177-07

Cited by 17 publications

(33 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Indeed, we measured an AmCyan-NtWLIM1 lifetime of 2.7560.07 nseconds, corresponding to a FRET efficiency of 2.8% (Fig. 5C), a value below the typical 5% significance threshold (Ciubotaru et al, 2007). Together, these data confirm the BiFC analyses and provide compelling evidence that NtWLIM1 self-associates along the actin cytoskeleton.…”

Section: Ntwlim1 Self-associates Along the Actin Cytoskeletonsupporting

confidence: 77%

Live cell imaging approaches reveal actin cytoskeleton-induced self-association of the actin-bundling protein WLIM1

Hoffmann

Moes

Dieterle

et al. 2013

Journal of Cell Science

View full text Add to dashboard Cite

Crosslinking of actin filaments into bundles is critical for the assembly/stabilization of specific cytoskeletal structures. Relatively little is known about the molecular mechanisms underlying actin bundle formation. The two LIM domain-containing (LIM) proteins define a novel and evolutionary-conserved family of actin bundlers whose actin-binding and -crosslinking activities primarily rely on their LIM domains. Using TIRF microscopy, we describe real-time formation of actin bundles induced by tobacco NtWLIM1 in vitro. We show that NtWLIM1 binds to single filaments and subsequently promotes their interaction and zippering into tight bundles of mixed polarity. NtWLIM1-induced bundles grew by both elongation of internal filaments and addition of preformed fragments at their extremities. Importantly, these data are highly consistent with the modes of bundle formation and growth observed in transgenic Arabidopsis plants expressing a GFP fused Arabidopsis AtWLIM1 protein. Using two complementary live cell imaging approaches, a close relationship between NtWLIM1 subcellular localization and self-association was established. Indeed, both BiFC and FLIM-FRET data revealed that, although unstable NtWLIM1 complexes can sporadically form in the cytosol, stable complexes concentrate along the actin cytoskeleton. Remarkably, the disruption of the actin cytoskeleton significantly impaired NtWLIM1 self-association. In addition, biochemical analyses support that F-actin facilitates the switch of purified recombinant NtWLIM1 from a monomeric to a di/oligomeric state. Based on our data we propose a model in which actin binding promotes the formation/stabilization of NtWLIM1 complexes, which in turn might drive the crosslinking of actin filaments.

show abstract

Section: Ntwlim1 Self-associates Along the Actin Cytoskeletonsupporting

confidence: 77%

Live cell imaging approaches reveal actin cytoskeleton-induced self-association of the actin-bundling protein WLIM1

Hoffmann

Moes

Dieterle

et al. 2013

Journal of Cell Science

View full text Add to dashboard Cite

show abstract

“…In addition, fluorescence resonance energy transfer techniques have recently been applied to examine the orientation of DNA strands within the synaptic complex. The data obtained from these experiments led the authors to favor a model in which the RSSs adopt a bent and crossed configuration in the synaptic complex, although an alternative model in which synaptic complexes containing RSSs in parallel and antiparallel configurations assemble with similar frequency could not be formally excluded (8). For most in vitro biochemical studies, the synaptic complex has been assembled by incubating the RAG proteins with a pair of oligonucleotide substrates, one containing a 12RSS, and one containing a 23RSS.…”

mentioning

confidence: 99%

Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation

Shlyakhtenko

Gilmore

Kriatchko

et al. 2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Two lymphoid cell-specific proteins, RAG1 and RAG2 (RAG), initiate V(D)J recombination by assembling a synaptic complex with recombination signal sequences (RSSs) abutting two different antigen receptor gene coding segments, and then introducing a DNA double strand break at the end of each RSS. Despite the biological importance of this system, the structure of the synaptic complex, and the RAG protein stoichiometry and arrangement of DNA within the synaptosome, remains poorly understood. Here we applied atomic force microscopy to directly visualize and characterize RAG synaptic complexes. We report that the pre-cleavage RAG synaptic complex contains about twice the protein content as a RAG complex bound to a single RSS, with a calculated mass consistent with a pair of RAG heterotetramers. In the synaptic complex, the RSSs are predominantly oriented in a sideby-side configuration with no DNA strand crossover. The mass of the synaptic complex, and the conditions under which it is formed in vitro, favors an association model of assembly in which isolated RAG-RSS complexes undergo synapsis mediated by RAG protein-protein interactions. The replacement of Mg 2؉ cations with Ca 2؉ leads to a dramatic change in protein stoichiometry for all RAG-RSS complexes, suggesting that the cation composition profoundly influences the type of complex assembled.To generate diverse surface antigen receptor molecules, developing lymphocytes undergo a series of site-specific DNA rearrangements to assemble functional antigen receptor genes from component gene segments (1). This DNA rearrangement process, known as V(D)J recombination, is initiated when two lymphoid cell-specific proteins, called RAG1 and RAG2, assemble a multiprotein synaptic complex with a pair of antigen receptor gene segments and subsequently introduce a DNA double strand break at the end of each gene segment (2). A recombination signal sequence (RSS) 3 that abuts each participating gene segment serves as the binding site of the RAG proteins and directs the location of DNA cleavage. Each RSS contains conserved heptamer and nonamer sequences that are separated by either 12 or 23 bp of DNA of more varied sequence (12RSS and 23RSS, respectively); efficient V(D)J recombination generally only occurs between two RSSs in which the lengths of DNA separating the heptamer and nonamer differ (the 12/23 rule). The RAG proteins mediate DNA cleavage via a nick-hairpin mechanism, breaking the DNA between the RSS heptamer and the coding segment; these reaction products are subsequently processed and repaired by the non-homologous end-joining pathway (1, 3).Previous studies suggest that RAG synaptic complexes are assembled through the stepwise binding of a 12RSS followed by the capture of a 23RSS (4 -6). In vitro biochemical studies suggest synapsis is mediated by a RAG1/2 heterotetramer, but there remains disagreement over the stoichiometry of RAG1 in these complexes (7). In addition, fluorescence resonance energy transfer techniques have recently been applied to examine the orientat...

show abstract

“…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%

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.

View full text Add to dashboard Cite

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...

show abstract

Fluorescence Resonance Energy Transfer Analysis of Recombination Signal Sequence Configuration in the RAG1/2 Synaptic Complex

Cited by 17 publications

References 42 publications

Live cell imaging approaches reveal actin cytoskeleton-induced self-association of the actin-bundling protein WLIM1

Live cell imaging approaches reveal actin cytoskeleton-induced self-association of the actin-bundling protein WLIM1

Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation

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

Contact Info

Product

Resources

About