Observation and Analysis of RAD51 Nucleation Dynamics at Single-Monomer Resolution

Subramanyam, Shyamal; Kinz-Thompson, Colin D.; Gonzalez, Ruben L.; Spies, Maria

doi:10.1016/bs.mie.2017.12.008

Cited by 17 publications

(15 citation statements)

References 56 publications

(93 reference statements)

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“…Notably, a surface-tethered RPA displayed essentially invariable, one state fluorescence with only very rare exertions into a dark state (Fig.S6a,b). Global analysis of normalized trajectories for the ssDNA-bound RPA-DBD-A MB543 and RPA-DBD-D MB543 was performed with ebFRET 27,28 . The number of trajectories and states in each experiment are summarized in the Supplemental Table S1 and Supplemental Dataset 4.…”

Section: Resultsmentioning

confidence: 99%

Dynamics and selective remodeling of the DNA-binding domains of RPA

Pokhrel

Caldwell

Corless

et al. 2019

Nat Struct Mol Biol

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Replication protein A (RPA) coordinates important DNA metabolic events by stabilizing single-strand DNA (ssDNA) intermediates, activating the DNA damage response and handing off ssDNA to appropriate downstream players. Six DNA binding domains (DBDs) in RPA promote high affinity binding to ssDNA yet also allow RPA displacement by lower affinity proteins. We generated fluorescent versions of Saccharomyces cerevisiae RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. We show that both DBD-A and DBD-D rapidly bind to and dissociate from ssDNA while RPA remains bound to ssDNA. The recombination mediator protein Rad52 selectively modulates the dynamics of DBD-D. These findings reveal how RPA interacting proteins with lower ssDNA binding affinities can access the occluded ssDNA and remodel individual DBDs to replace RPA.

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Section: Resultsmentioning

confidence: 99%

Dynamics and selective remodeling of the DNA-binding domains of RPA

Pokhrel

Caldwell

Corless

et al. 2019

Nat Struct Mol Biol

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117

143

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“…The RAD51-ssDNA filament is over-stretched relative to naked and RPA-coated ssDNA ( 35 ). The extent of ssDNA extension serves as a convenient reporter for RAD51 filament assembly and extension ( 33 , 36 ).…”

Section: Resultsmentioning

confidence: 99%

RADX condenses single-stranded DNA to antagonize RAD51 loading

Zhang

Schaub

Finkelstein

2020

Nucleic Acids Research

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Abstract RADX is a mammalian single-stranded DNA-binding protein that stabilizes telomeres and stalled replication forks. Cellular biology studies have shown that the balance between RADX and Replication Protein A (RPA) is critical for DNA replication integrity. RADX is also a negative regulator of RAD51-mediated homologous recombination at stalled forks. However, the mechanism of RADX acting on DNA and its interactions with RPA and RAD51 are enigmatic. Using single-molecule imaging of the key proteins in vitro, we reveal that RADX condenses ssDNA filaments, even when the ssDNA is coated with RPA at physiological protein ratios. RADX compacts RPA-coated ssDNA filaments via higher-order assemblies that can capture ssDNA in trans. Furthermore, RADX blocks RPA displacement by RAD51 and prevents RAD51 loading on ssDNA. Our results indicate that RADX is an ssDNA condensation protein that inhibits RAD51 filament formation and may antagonize other ssDNA-binding proteins on RPA-coated ssDNA.

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“…Moreover, transitions between different fluorescence states persisted during the last segment of the experiment suggesting that they truly reflect the conformational dynamics of individual RPA-ssDNA complexes. Global analysis of normalized trajectories for the ssDNA-bound RPA-DBD-A MB543 and RPA-DBD-D MB543 was performed with ebFRET, a program that determines the number of states using an Empirical Bayesian method to generate Hidden Markov Models 26,27 . The number of trajectories and states in each experiment are summarized in the Supplemental Table S1 and Fig.…”

Section: Differential Effects Of Ssdna Length On Dbd Conformationsmentioning

confidence: 99%

Dynamics and Selective Remodeling of the DNA Binding Domains of RPA

Pokhrel

Caldwell

Corless

et al. 2018

Preprint

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Replication protein A (RPA) coordinates important DNA metabolic events by stabilizing singlestrand DNA (ssDNA) intermediates, activating the DNA damage response, and handing off ssDNA to appropriate downstream players. Six DNA binding domains (DBDs) in RPA promote high affinity binding to ssDNA, but also allow RPA displacement by lower affinity proteins. We have made fluorescent versions of RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. We show that both DBD-A and DBD-D rapidly bind to and dissociate from ssDNA, while RPA as a whole remains bound to ssDNA.The recombination mediator protein Rad52 selectively modulates the dynamics of DBD-D. This demonstrates how RPA interacting proteins, with lower ssDNA binding affinity, can access the occluded ssDNA and remodel individual DBDs to replace RPA. Main Text:In every eukaryotic cell, RPA binds to transiently exposed ssDNA and serves as a hub protein to coordinate essential DNA metabolic processes including replication, recombination, repair, and telomere maintenance 1,2 . Cellular functions of RPA rely on its high affinity ssDNA binding, its ability to physically interact with over two dozen DNA processing enzymes, and to correctly position these enzymes on complex DNA structures. The precise mechanisms through which RPA functions in many contexts; and more importantly, how it differentiates between multiple DNA metabolic events (DNA replication, repair or recombination) is a long-standing puzzle 1,3 .RPA is heterotrimeric, flexible, and modular in structure. It is composed of three subunits:RPA70, RPA32 and RPA14 (Figs.1a, b). The subunits harbor six 3 oligonucleotide/oligosaccharide binding folds (OB-folds; labeled A through F). We refer to the DNA binding OB-folds as DNA binding domains (DBDs; Fig. 1a). RPA binds to ssDNA with high, sub-nanomolar affinity, but can be displaced by DNA binding proteins with much lower DNA binding affinity. Recent studies have suggested that the RPA-ssDNA complex is relatively dynamic 4,5,6, positing a selective dissociative mechanism where not all DBDs are stably bound to the DNA at the same time and microscopic dissociation of individual DNA binding domains occurs.In all existing models for RPA function, DBDs A and B are assigned as high affinity binding domains. Purified DBD-A, DBD-B and DBD-A/DBD-B constructs bind ssDNA with KD values 2 µM, 20 µM and 50 nM, respectively 7-9 . The trimerization core made up of DBD-C (RPA70), DBD-D (RPA32) and DBD-E (RPA14) is considered to have a weaker ssDNA binding affinity (Kd>5μM) 10 . Additionally, mutational analysis of individual aromatic residues that interact with the ssDNA in either DBD-C or DBD-D show minimal perturbations on ssDNA binding affinity 6 . Paradoxically, in the recently solved crystal structure of the RPA-ssDNA complex (Fig. 1b), the interactions of all four DBD's with ssDNA are similar with DBD-C having more contacts with ssDNA bases than DBD-A, DBD-B or DBD-D 11 . Thus, the exact nature of the contributions from...

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Observation and Analysis of RAD51 Nucleation Dynamics at Single-Monomer Resolution

Cited by 17 publications

References 56 publications

Dynamics and selective remodeling of the DNA-binding domains of RPA

Dynamics and selective remodeling of the DNA-binding domains of RPA

RADX condenses single-stranded DNA to antagonize RAD51 loading

Dynamics and Selective Remodeling of the DNA Binding Domains of RPA

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