The Active DNA-PK Holoenzyme Occupies a Tensed State in a Staggered Synaptic Complex

2020 ◽  
Author(s):  
Morgan Hepburn ◽  
Daniel J. Saltzberg ◽  
Linda Lee ◽  
Shujuan Fang ◽  
Claire Atkinson ◽  
...  
Keyword(s):  
Synaptic Complex

scholarly journals Site-Directed Mutagenesis Studies of Tn5 Transposase Residues Involved in Synaptic Complex Formation

2007 ◽  
Vol 189 (20) ◽  
pp. 7436-7441 ◽  
Author(s):  
Soheila Vaezeslami ◽  
Rachel Sterling ◽  
William S. Reznikoff
Keyword(s):  
Complex Formation ◽  
Catalytic Reactions ◽  
Site Directed Mutagenesis ◽  
Dna Interactions ◽  
Synaptic Complex ◽  
Biochemical Assays ◽  
Protein Dna Interactions ◽  
End Sequences ◽  
Tn5 Transposase ◽  
Cocrystal Structure

ABSTRACT Transposition (the movement of discrete segments of DNA, resulting in rearrangement of genomic DNA) initiates when transposase forms a dimeric DNA-protein synaptic complex with transposon DNA end sequences. The synaptic complex is a prerequisite for catalytic reactions that occur during the transposition process. The transposase-DNA interactions involved in the synaptic complex have been of great interest. Here we undertook a study to verify the protein-DNA interactions that lead to synapsis in the Tn5 system. Specifically, we studied (i) Arg342, Glu344, and Asn348 and (ii) Ser438, Lys439, and Ser445, which, based on the previously published cocrystal structure of Tn5 transposase bound to a precleaved transposon end sequence, make cis and trans contacts with transposon end sequence DNA, respectively. By using genetic and biochemical assays, we showed that in all cases except one, each of these residues plays an important role in synaptic complex formation, as predicted by the cocrystal structure.

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

2016 ◽  
Vol 113 (42) ◽  
pp. 11853-11858 ◽  
Author(s):  
Jennifer Zagelbaum ◽  
Noriko Shimazaki ◽  
Zitadel Anne Esguerra ◽  
Go Watanabe ◽  
Michael R. Lieber ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Conformational Changes ◽  
Signal Sequence ◽  
High Mobility ◽  
Time Analysis ◽  
Complex Activity ◽  
Synaptic Complex ◽  
Single Molecule Fret ◽  
Real Time Analysis

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.

scholarly journals Rous Sarcoma Virus Synaptic Complex Capable of Concerted Integration Is Kinetically Trapped by Human Immunodeficiency Virus Integrase Strand Transfer Inhibitors

2014 ◽  
Vol 289 (28) ◽  
pp. 19648-19658 ◽  
Author(s):  
Krishan K. Pandey ◽  
Sibes Bera ◽  
Sergey Korolev ◽  
Mary Campbell ◽  
Zhiqi Yin ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rous Sarcoma Virus ◽  
Strand Transfer ◽  
Synaptic Complex ◽  
Rous Sarcoma ◽  
Immunodeficiency Virus ◽  
Integrase Strand Transfer Inhibitors ◽  
Sarcoma Virus

scholarly journals Architecture of the Hin Synaptic Complex during Recombination

Cell ◽  
2004 ◽  
Vol 119 (1) ◽  
pp. 33-45 ◽  
Author(s):  
Gautam Dhar ◽  
Erin R. Sanders ◽  
Reid C. Johnson
Keyword(s):  
Synaptic Complex

scholarly journals RAG-Heptamer Interaction in the Synaptic Complex Is a Crucial Biochemical Checkpoint for the 12/23 Recombination Rule

2007 ◽  
Vol 283 (8) ◽  
pp. 4877-4885 ◽  
Author(s):  
Tadashi Nishihara ◽  
Fumikiyo Nagawa ◽  
Takeshi Imai ◽  
Hitoshi Sakano
Keyword(s):  
Synaptic Complex

Structure of a NHEJ Polymerase-Mediated DNA Synaptic Complex

Science ◽  
2007 ◽  
Vol 318 (5849) ◽  
pp. 456-459 ◽  
Author(s):  
N. C. Brissett ◽  
R. S. Pitcher ◽  
R. Juarez ◽  
A. J. Picher ◽  
A. J. Green ◽  
...  
Keyword(s):  
Synaptic Complex

A novel type of microglomerular synaptic complex in the polarization vision pathway of the locust brain

2007 ◽  
Vol 506 (2) ◽  
pp. 288-300 ◽  
Author(s):  
Ulrike Träger ◽  
Robert Wagner ◽  
Bernhard Bausenwein ◽  
Uwe Homberg
Keyword(s):  
Polarization Vision ◽  
Synaptic Complex

scholarly journals Assembly, translocation, and activation of XerCD-difrecombination by FtsK translocase analyzed in real-time by FRET and two-color tethered fluorophore motion

2015 ◽  
Vol 112 (37) ◽  
pp. E5133-E5141 ◽  
Author(s):  
Peter F. J. May ◽  
Pawel Zawadzki ◽  
David J. Sherratt ◽  
Achillefs N. Kapanidis ◽  
Lidia K. Arciszewska
Keyword(s):  
Real Time ◽  
Bacterial Chromosome ◽  
Dna Loops ◽  
Synaptic Complex ◽  
Dna Molecule ◽  
Subsequent Activation ◽  
Replication Terminus

The FtsK dsDNA translocase functions in bacterial chromosome unlinking by activating XerCD-difrecombination in the replication terminus region. To analyze FtsK assembly and translocation, and the subsequent activation of XerCD-difrecombination, we extended the tethered fluorophore motion technique, using two spectrally distinct fluorophores to monitor two effective lengths along the same tethered DNA molecule. We observed that FtsK assembled stepwise on DNA into a single hexamer, and began translocation rapidly (∼0.25 s). Without extruding DNA loops, single FtsK hexamers approached XerCD-difand resided there for ∼0.5 s irrespective of whether XerCD-difwas synapsed or unsynapsed. FtsK then dissociated, rather than reversing. Infrequently, FtsK activated XerCD-difrecombination when it encountered a preformed synaptic complex, and dissociated before the completion of recombination, consistent with each FtsK–XerCD-difencounter activating only one round of recombination.

scholarly journals Membrane-associated guanylate kinase scaffolds organize a horizontal cell synaptic complex restricted to invaginating contacts with photoreceptors

2016 ◽  
Vol 525 (4) ◽  
pp. 850-867 ◽  
Author(s):  
Alejandro Vila ◽  
Christopher M. Whitaker ◽  
John O'Brien
Keyword(s):  
Horizontal Cell ◽  
Guanylate Kinase ◽  
Synaptic Complex

scholarly journals The novel synaptogenic protein Farp1 links postsynaptic cytoskeletal dynamics and transsynaptic organization

2012 ◽  
Vol 199 (6) ◽  
pp. 985-1001 ◽  
Author(s):  
Lucas Cheadle ◽  
Thomas Biederer
Keyword(s):  
Synapse Formation ◽  
Synaptic Membranes ◽  
Synapse Development ◽  
The Novel ◽  
Spine Density ◽  
Actin Assembly ◽  
Spine Morphology ◽  
Synaptic Complex ◽  
Cytoskeletal Dynamics ◽  
Retrograde Signal

Synaptic adhesion organizes synapses, yet the signaling pathways that drive and integrate synapse development remain incompletely understood. We screened for regulators of these processes by proteomically analyzing synaptic membranes lacking the synaptogenic adhesion molecule SynCAM 1. This identified FERM, Rho/ArhGEF, and Pleckstrin domain protein 1 (Farp1) as strongly reduced in SynCAM 1 knockout mice. Farp1 regulates dendritic filopodial dynamics in immature neurons, indicating roles in synapse formation. Later in development, Farp1 is postsynaptic and its 4.1 protein/ezrin/radixin/moesin (FERM) domain binds SynCAM 1, assembling a synaptic complex. Farp1 increases synapse number and modulates spine morphology, and SynCAM 1 requires Farp1 for promoting spines. In turn, SynCAM 1 loss reduces the ability of Farp1 to elevate spine density. Mechanistically, Farp1 activates the GTPase Rac1 in spines downstream of SynCAM 1 clustering, and promotes F-actin assembly. Farp1 furthermore triggers a retrograde signal regulating active zone composition via SynCAM 1. These results reveal a postsynaptic signaling pathway that engages transsynaptic interactions to coordinate synapse development.

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