Quantitative distance information on protein-DNA complexes determined in polyacrylamide gels by fluorescence resonance energy transfer

Lorenz, Michael G.; Diekmann, Stephan

doi:10.1002/1522-2683()22:6<990::aid-elps990>3.0.co;2-x

Cited by 19 publications

(8 citation statements)

References 31 publications

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“…To facilitate construction of a structural model using the Int bridging patterns determined in the companion paper (16), we used in-gel FRET to determine the apparent distances between several positions within the excisive recombination HJ intermediate complex (19)(20)(21)(22). The efficiencies of energy transfer between donor and acceptor fluorophores were determined by comparing the extent of donor fluorescence quenching in complexes, either with or without the acceptor, as described previously (20).…”

Section: Resultsmentioning

confidence: 99%

“…The crystal structure of IHF complexed with its cognate DNA target, H′, was a useful source of R, because this same complex is an integral structural element in both the integrative and excisive recombination pathways; it has also been the subject of several FRET studies in solution and in gels (19,25,26). We therefore positioned donor and acceptor fluorophores near the two termini of an IHF-induced H′ DNA loop and measured the forward and reverse FRET efficiencies (see Fig.…”

Section: Resultsmentioning

confidence: 99%

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Nucleoprotein architectures regulating the directionality of viral integration and excision

Seah

Warren

Tong

et al. 2014

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

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The virally encoded site-specific recombinase Int collaborates with its accessory DNA bending proteins IHF, Xis, and Fis to assemble two distinct, very large, nucleoprotein complexes that carry out either integrative or excisive recombination along regulated and essentially unidirectional pathways. The core of each complex consists of a tetramer of Integrase protein (Int), which is a heterobivalent DNA binding protein that binds and bridges a core-type DNA site (where strand cleavage and ligation are executed), and a distal arm-type site, that is brought within range by one or more DNA bending proteins. The recent determination of the patterns of these Int bridges has made it possible to think realistically about the global architecture of the recombinogenic complexes. Here, we combined the previously determined Int bridging patterns with in-gel FRET experiments and in silico modeling to characterize and differentiate the two 400-kDa multiprotein Holiday junction recombination intermediates formed during λ integration and excision. The results lead to architectural models that explain how integration and excision are regulated in λ site-specific recombination. Our confidence in the basic features of these architectures is based on the redundancy and self-consistency of the underlying data from two very different experimental approaches to establish bridging interactions, a set of strategic intracomplex distances from FRET experiments, and the model's ability to explain key aspects of the integrative and excisive recombination pathways, such as topological changes, the mechanism of capturing attB, and the features of asymmetry and flexibility within the complexes.regulation of directionality | topology | recombinogenic architectures | molecular machines H igh-precision DNA transactions responsible for a variety of fundamental processes are typically promoted and modulated by large multiprotein machines that use cooperative interactions and involve DNA bending and/or wrapping. One well-studied example is the tightly regulated and highly directional site-specific recombination by which bacteriophage λ inserts and excises its DNA into and out of the Escherichia coli host chromosome, using the phage attP and the bacterial attB DNA sequences for integration and the resulting junction sequences, attL and attR, for excision. These reactions are catalyzed by the phage-encoded Integrase protein (Int), the founding member of the tyrosine recombinase family of site-specific recombinases (1). In addition to mediating integration and excision of viral genomes, members of this family function in a variety of other cellular processes including chromosome segregation, gene regulation, and conjugative transposition (2).Int has three well-characterized domains: an N-terminal DNA-binding domain (NTD), a central core-binding domain (CB), and a C-terminal catalytic domain (CAT). The CB and CAT domains (referred to here as the CTD) are together responsible for binding to the core-type DNA sequences where strand exchange and ligation t...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Nucleoprotein architectures regulating the directionality of viral integration and excision

Seah

Warren

Tong

et al. 2014

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

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“…Quenching in the PC correlates qualitatively with FRET in solution (HN4 Ͼ HN2 Ͼ HH2) and was reproducible. Values of quenching in the gel were lower than efficiencies of energy transfer in solution at least in part because acrylamide is a fluorescence quencher that reduces the Förster radius (25).…”

Section: Vol 27 2007 Fret Analysis Of Rss Configuration 4751mentioning

confidence: 99%

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

Ciubotaru

Kriatchko

Swanson

et al. 2007

Molecular and Cellular Biology

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The assembly of the gene segments coding for the variable portions of lymphocyte antigen receptors (immunoglobulins and T-cell receptors) occurs via a somatic recombination reaction known as V(D)J recombination. This process is mediated by two lymphoid cell-specific factors, RAG1 and RAG2 (recombination-activating gene products), that perform DNA cleavage at a pair of recombination signal sequences (RSSs) flanking the coding segments to be joined. RSSs consist of short palindromic heptamer and A/T-rich nonamer elements separated by a less well conserved spacer of 12 or 23 bp (12-RSS and 23-RSS). In vivo, recombination occurs most efficiently with a 12/23 RSS pair, a phenomenon referred to as the 12/23 rule.Under appropriate conditions in vitro, the RAG proteins together with high-mobility-group protein HMGB1 or HMGB2 perform coupled DNA cleavage of RSS substrates in accordance with the 12/23 rule (16). This process is thought to be initiated by the binding of the RAG recombinase to one RSS, followed by the capture of a second RSS, thus assembling the synaptic, or paired, complex (PC) (22, 28). HMGB1 and HMGB2 proteins play critical roles in vitro in facilitating the binding of the RAG proteins to the 23-RSS and the formation of the PC (15, 16), activities thought to rely on their ability to recognize bent or distorted DNA structures (3, 7). DNA cleavage takes place in two steps: a nick is first introduced between the heptamer and the coding DNA, and the 3Ј hydroxyl group thus liberated then attacks the other strand of the duplex to generate a covalently sealed hairpin coding end and a blunt signal end (16). Nicking can occur before or after synapsis, but hairpin formation occurs coordinately at the two RSSs within the PC (15). The PC is thus a critical intermediate in which the recombining partners are chosen and DNA double-strand breaks are made.Numerous studies have identified functionally important residues and domains within the catalytically essential, or core, region of RAG1 (10): an N-terminal nonamer binding domain that interacts with the RSS nonamer, a central domain that interacts with RAG2 and the heptamer and contains two of three acidic residues thought to contribute to the RAG active site, and a C-terminal domain with dimerization and nonspecific DNA binding activity and the third active-site residue. Less is known about the RAG2 core region.The structure of the PC is poorly understood. The PC is thought to contain two (37) or more than two (28) RAG1 monomers, two monomers of RAG2 (28,37), and an unknown number of HMGB1 or HMGB2 subunits. The nonamer binding and catalytic domains that interact with a particular RSS are contributed by different RAG1 monomers (36). This and the dependence of hairpin formation upon synapsis suggest careful coordination between the catalytic events at the two RSSs, a possibility supported by the finding that nicking at one RSS is required for hairpin formation at the partner RSS (41).Very little is known about the structure of the two DNA molecules in the PC. RAG1/2 an...

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“…Donor and acceptor fluorophores can be incorporated site-specifically into nucleic acids and recombinant proteins (e.g. via unique cysteine residues [7]), and resulting FRET signals can be monitored either in gelbased assays [8] or in solution, in ensemble or at the singlemolecule level. Ensemble fluorescence measurements [9] are obtained from a vast number of molecules and therefore represent an average fluorescence signal arising from multiple species.…”

Section: Analysing Rnaps (Rna Polymerases) With Lightmentioning

confidence: 99%

FRET (fluorescence resonance energy transfer) sheds light on transcription

Grohmann

Klose

Fielden

et al. 2011

Biochemical Society Transactions

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The complex organization of the transcription machinery has been revealed mainly by biochemical and crystallographic studies. X-ray structures describe RNA polymerases and transcription complexes on an atomic level, but fail to portray their dynamic nature. The use of fluorescence techniques has made it possible to add a new layer of information to our understanding of transcription by providing details about the structural rearrangement of mobile elements and the network of interactions within transcription complexes in solution and in real-time.

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Quantitative distance information on protein-DNA complexes determined in polyacrylamide gels by fluorescence resonance energy transfer

Cited by 19 publications

References 31 publications

Nucleoprotein architectures regulating the directionality of viral integration and excision

Nucleoprotein architectures regulating the directionality of viral integration and excision

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

FRET (fluorescence resonance energy transfer) sheds light on transcription

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