Dataset Information

Fluorescence resonance energy transfer analysis of recombination signal sequence configuration in the RAG1/2 synaptic complex.

ABSTRACT: A critical step in V(D)J recombination is the synapsis of complementary (12/23) recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins to generate the paired complex (PC). Using a facilitated ligation assay and substrates that vary the helical phasing of the RSSs, we provide evidence that one particular geometric configuration of the RSSs is favored in the PC. To investigate this configuration further, we used fluorescent resonance energy transfer (FRET) to detect the synapsis of fluorescently labeled RSS oligonucleotides. FRET requires an appropriate 12/23 RSS pair, a divalent metal ion, and high-mobility-group protein HMGB1 or HMGB2. Energy transfer between the RSSs was detected with all 12/23 RSS end positions of the fluorescent probes but was not detected when probes were placed on the two ends of the same RSS. Energy transfer was confirmed to originate from the PC by using an in-gel FRET assay. The results argue against a unique planar configuration of the RSSs in the PC and are most easily accommodated by models in which synapsed 12- and 23-RSSs are bent and cross one another, with implications for the organization of the RAG proteins and the DNA substrates at the time of cleavage.

SUBMITTER: Ciubotaru M

PROVIDER: S-EPMC1951485 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Fluorescence resonance energy transfer analysis of recombination signal sequence configuration in the RAG1/2 synaptic complex.

Ciubotaru Mihai M Kriatchko Aleksei N AN Swanson Patrick C PC Bright Frank V FV Schatz David G DG

Molecular and cellular biology 20070430 13

A critical step in V(D)J recombination is the synapsis of complementary (12/23) recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins to generate the paired complex (PC). Using a facilitated ligation assay and substrates that vary the helical phasing of the RSSs, we provide evidence that one particular geometric configuration of the RSSs is favored in the PC. To investigate this configuration further, we used fluorescent resonance energy transfer (FRET) to detect the synapsis of fl ...[more]

PMID: 17470556

Similar Datasets

Project description:Conjugated polydiacetylene (PDA) possessing stimuli-responsive properties has been intensively investigated for developing efficient sensors. We report here fluorescence resonance energy transfer (FRET) in liposomes synthesized using different molar ratios of dansyl-tagged diacetylene and diacetylene-carboxylic acid monomers. Photopolymerization of diacetylene resulted in cross-linked PDA liposomes. We used steady-state electronic absorption, emission, and fluorescence anisotropy (FA) analysis to characterize the thermal-induced FRET between dansyl fluorophores (donor) and PDA (acceptor). We found that the monomer ratio of acceptor to donor ( R ad) and length of linkers (functional part that connects dansyl fluorophores to the diacetylene group in the monomer) strongly affected FRET. For R ad = 10 000, the acceptor emission intensity was amplified by more than 18 times when the liposome solution was heated from 298 to 338 K. A decrease in R ad resulted in diminished acceptor emission amplification. This was primarily attributed to lower FRET efficiency between donors and acceptors and a higher background signal. We also found that the FRET amplification of PDA emissions after heating the solution was much higher when dansyl was linked to diacetylene through longer and flexible linkers than through shorter linkers. We attributed this to insertion of dansyl in the bilayer of the liposomes, which led to an increased dansyl quantum yield and a higher interaction of multiple acceptors with limited available donors. This was not the case for shorter and more rigid linkers where PDA amplification was much smaller. The present studies aim at enhancing our understanding of FRET between fluorophores and PDA-based conjugated liposomes. Furthermore, receptor tagged onto PDA liposomes can interact with ligands present on proteins, enzymes, and cells, which will produce emission sensing signal. Therefore, using the present approach, there exist opportunities for designing FRET-based highly sensitive and selective chemical and biochemical sensors.

Dataset Information

Fluorescence resonance energy transfer analysis of recombination signal sequence configuration in the RAG1/2 synaptic complex.

Publications

Fluorescence resonance energy transfer analysis of recombination signal sequence configuration in the RAG1/2 synaptic complex.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets