scholarly journals Dynamics and Selective Remodeling of the DNA Binding Domains of RPA

2018 ◽  
Author(s):  
Nilisha Pokhrel ◽  
Colleen C. Caldwell ◽  
Elliot I. Corless ◽  
Emma A. Tillison ◽  
Joseph Tibbs ◽  
...  
Keyword(s):  
Dna Binding ◽  
Conformational Dynamics ◽  
Protein A ◽  
Interacting Proteins ◽  
Ssdna Binding ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Mediator Protein ◽  
Damage Response ◽  
The Individual

AbstractReplication 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, 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.One Sentence SummaryThe choreography of binding and rearrangement of the individual domains of RPA during homologous recombination is revealed.

scholarly journals Targeting the OB-Folds of Replication Protein A with Small Molecules

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Victor J. Anciano Granadillo ◽  
Jennifer N. Earley ◽  
Sarah C. Shuck ◽  
Millie M. Georgiadis ◽  
Richard W. Fitch ◽  
...  
Keyword(s):  
Dna Binding ◽  
Protein A ◽  
Replication Protein A ◽  
Dna Interaction ◽  
Replication Protein ◽  
Ssdna Binding ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Target Binding

Replication protein A (RPA) is the main eukaryotic single-strand (ss) DNA-binding protein involved in DNA replication and repair. We have identified and developed two classes of small molecule inhibitors (SMIs) that showin vitroinhibition of the RPA-DNA interaction. We present further characterization of these SMIs with respect to their target binding, mechanism of action, and specificity. Both reversible and irreversible modes of inhibition are observed for the different classes of SMIs with one class found to specifically interact with DNA-binding domains A and B (DBD-A/B) of RPA. In comparison with other oligonucleotide/oligosaccharide binding-fold (OB-fold) containing ssDNA-binding proteins, one class of SMIs displayed specificity for the RPA protein. Together these data demonstrate that the specific targeting of a protein-DNA interaction can be exploited towards interrogating the cellular activity of RPA as well as increasing the efficacy of DNA-damaging chemotherapeutics used in cancer treatment.

scholarly journals Identification of the DNA-Binding Domains of Human Replication Protein A That Recognize G-Quadruplex DNA

2011 ◽  
Vol 2011 ◽  
pp. 1-14 ◽  
Author(s):  
Aishwarya Prakash ◽  
Amarnath Natarajan ◽  
Luis A. Marky ◽  
Michel M. Ouellette ◽  
Gloria E. O. Borgstahl
Keyword(s):  
Dna Binding ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Binding Studies ◽  
Quadruplex Dna ◽  
Ssdna Binding ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
G Quadruplex

Replication protein A (RPA), a key player in DNA metabolism, has 6 single-stranded DNA-(ssDNA-) binding domains (DBDs) A-F. SELEX experiments with the DBDs-C, -D, and -E retrieve a 20-nt G-quadruplex forming sequence. Binding studies show that RPA-DE binds preferentially to the G-quadruplex DNA, a unique preference not observed with other RPA constructs. Circular dichroism experiments show that RPA-CDE-core can unfold the G-quadruplex while RPA-DE stabilizes it. Binding studies show that RPA-C binds pyrimidine- and purine-rich sequences similarly. This difference between RPA-C and RPA-DE binding was also indicated by the inability of RPA-CDE-core to unfold an oligonucleotide containing a TC-region 5′ to the G-quadruplex. Molecular modeling studies of RPA-DE and telomere-binding proteins Pot1 and Stn1 reveal structural similarities between the proteins and illuminate potential DNA-binding sites for RPA-DE and Stn1. These data indicate that DBDs of RPA have different ssDNA recognition properties.

scholarly journals Independent and Coordinated Functions of Replication Protein A Tandem High Affinity Single-stranded DNA Binding Domains

2003 ◽  
Vol 278 (42) ◽  
pp. 41077-41082 ◽  
Author(s):  
Alphonse I. Arunkumar ◽  
Melissa E. Stauffer ◽  
Elena Bochkareva ◽  
Alexey Bochkarev ◽  
Walter J. Chazin
Keyword(s):  
Dna Binding ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Single Stranded Dna ◽  
Dna Binding Domains ◽  
High Affinity ◽  
Binding Domains

scholarly journals Understanding nuclear receptor form and function using structural biology

2013 ◽  
Vol 51 (3) ◽  
pp. T1-T21 ◽  
Author(s):  
Fraydoon Rastinejad ◽  
Pengxiang Huang ◽  
Vikas Chandra ◽  
Sepideh Khorasanizadeh
Keyword(s):  
Dna Binding ◽  
Structural Biology ◽  
Therapeutic Potential ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Dna Binding Domains ◽  
Form And Function ◽  
Receptor Complexes ◽  
Binding Domains ◽  
The Individual

Nuclear receptors (NRs) are a major transcription factor family whose members selectively bind small-molecule lipophilic ligands and transduce those signals into specific changes in gene programs. For over two decades, structural biology efforts were focused exclusively on the individual ligand-binding domains (LBDs) or DNA-binding domains of NRs. These analyses revealed the basis for both ligand and DNA binding and also revealed receptor conformations representing both the activated and repressed states. Additionally, crystallographic studies explained how NR LBD surfaces recognize discrete portions of transcriptional coregulators. The many structural snapshots of LBDs have also guided the development of synthetic ligands with therapeutic potential. Yet, the exclusive structural focus on isolated NR domains has made it difficult to conceptualize how all the NR polypeptide segments are coordinated physically and functionally in the context of receptor quaternary architectures. Newly emerged crystal structures of the peroxisome proliferator-activated receptor-γ–retinoid X receptor α (PPARγ–RXRα) heterodimer and hepatocyte nuclear factor (HNF)-4α homodimer have recently revealed the higher order organizations of these receptor complexes on DNA, as well as the complexity and uniqueness of their domain–domain interfaces. These emerging structural advances promise to better explain how signals in one domain can be allosterically transmitted to distal receptor domains, also providing much better frameworks for guiding future drug discovery efforts.

scholarly journals Slow, heterogeneous NFκB single molecule conformational dynamics and implications for function

2021 ◽  
Author(s):  
Wei Chen ◽  
Wei Liu ◽  
Peter Wolynes ◽  
Elizabeth A. Komives
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Electrostatic Interactions ◽  
Bound States ◽  
Conformational Dynamics ◽  
Dna Binding Domains ◽  
Single Molecule Fret ◽  
Binding Domains ◽  
Lower Amplitude ◽  
Slow Motions

The transcription factor NFκB (RelA-p50) is a multidomain protein that binds DNA and its inhibitor, IκBα with apparently different conformations. We used single-molecule FRET to characterize the interdomain motions of the N-terminal DNA-binding domains in the free protein and also in various bound states. Several surprising results emerged from this study. First, the domains moved with respect to each other on several widely different timescales from hundreds of milliseconds to minutes. The free NFκB displayed stochastic motions leading to a broad distribution of states, ranging from very low-FRET states to high-FRET states. Varying the ionic strength altered the slow motions suggesting that they may be due to different weak electrostatic interactions between the domains creating a rugged energy landscape. Third, the DNA-binding domains continued to be mobile even when the protein was bound to its cognate DNA, but in this case the majority of the states were either high-FRET, a state expected from the available x-ray structures, or low-FRET, a state consistent with one of the DNA-binding domains dissociated. The fluctuations of the DNA-bound states were of lower amplitude and slightly faster frequency. Fourth, the inhibitor, IκBα freezes the domains into a low-FRET state, expected to be incapable of binding DNA. Neutralization of five acidic residues in the IκBα PEST sequence, which was previously shown to impair IκBαs ability to strip NFκB from the DNA, also impaired its ability to freeze the domains into a low-FRET state indicating that the freezing of motions of the DNA-binding domains is essential for efficient molecular stripping.

Letter to the Editor:1H,13C and15N Assignments of Single-Stranded DNA Binding Domains from the 70 kDa Subunit of Human Replication Protein A

2004 ◽  
Vol 28 (2) ◽  
pp. 195-196 ◽  
Author(s):  
Shibani Bhattacharya ◽  
Alphonse I. Arunkumar ◽  
Shannon L. Sullivan ◽  
Maria-Victoria Botuyan ◽  
Cheryl H. Arrowsmith ◽  
...  
Keyword(s):  
Dna Binding ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Single Stranded Dna ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Letter To The Editor

scholarly journals Mechanistic Heterogeneity in Site Recognition by the Structurally Homologous DNA-binding Domains of the ETS Family Transcription Factors Ets-1 and PU.1

2014 ◽  
Vol 289 (31) ◽  
pp. 21605-21616 ◽  
Author(s):  
Shuo Wang ◽  
Miles H. Linde ◽  
Manoj Munde ◽  
Victor D. Carvalho ◽  
W. David Wilson ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Ets Family ◽  
Site Recognition
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