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 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 Stabilization of a G-Quadruplex from Unfolding by Replication Protein A Using Potassium and the Porphyrin TMPyP4

2011 ◽  
Vol 2011 ◽  
pp. 1-13 ◽  
Author(s):  
Aishwarya Prakash ◽  
Fabien Kieken ◽  
Luis A. Marky ◽  
Gloria E. O. Borgstahl
Keyword(s):  
Dna Replication ◽  
Protein A ◽  
Thermal Stabilization ◽  
Replication Protein A ◽  
Replication Protein ◽  
Unique Problem ◽  
Ssdna Binding ◽  
Binding Domains ◽  
G Quadruplex ◽  
Stabilization Effect

Replication protein A (RPA) plays an essential role in DNA replication by binding and unfolding non-canonical single-stranded DNA (ssDNA) structures. Of the six RPA ssDNA binding domains (labeled A-F), RPA-CDE selectively binds a G-quadruplex forming sequence (5′-TAGGGGAAGGGTTGGAGTGGGTT-3′called Gq23). In K+, Gq23 forms a mixed parallel/antiparallel conformation, and in Na+Gq23 has a less stable (TMlowered by ∼20∘C), antiparallel conformation. Gq23 is intramolecular and 1D NMR confirms a stable G-quadruplex structure in K+. Full-length RPA and RPA-CDE-core can bind and unfold the Na+form of Gq23 very efficiently, but complete unfolding is not observed with the K+form. Studies with G-quadruplex ligands, indicate that TMPyP4 has a thermal stabilization effect on Gq23 in K+, and inhibits complete unfolding by RPA and RPA-CDE-core. Overall these data indicate that G-quadruplexes present a unique problem for RPA to unfold and ligands, such as TMPyP4, could possibly hinder DNA replication by blocking unfolding by RPA.

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

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 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 Identification and Characterization of the Fourth Single-Stranded-DNA Binding Domain of Replication Protein A

1998 ◽  
Vol 18 (12) ◽  
pp. 7225-7234 ◽  
Author(s):  
Steven J. Brill ◽  
Suzanne Bastin-Shanower
Keyword(s):  
Protein A ◽  
Replication Protein A ◽  
Binding Domain ◽  
Replication Protein ◽  
Binding Motif ◽  
Terminal Portion ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Amino Terminal ◽  
Binding Domains

ABSTRACT Replication protein A (RPA), the heterotrimeric single-stranded-DNA (ssDNA) binding protein (SSB) of eukaryotes, contains two homologous ssDNA binding domains (A and B) in its largest subunit, RPA1, and a third domain in its second-largest subunit, RPA2. Here we report that Saccharomyces cerevisiae RPA1 contains a previously undetected ssDNA binding domain (domain C) lying in tandem with domains A and B. The carboxy-terminal portion of domain C shows sequence similarity to domains A and B and to the region of RPA2 that binds ssDNA (domain D). The aromatic residues in domains A and B that are known to stack with the ssDNA bases are conserved in domain C, and as in domain A, one of these is required for viability in yeast. Interestingly, the amino-terminal portion of domain C contains a putative Cys4-type zinc-binding motif similar to that of another prokaryotic SSB, T4 gp32. We demonstrate that the ssDNA binding activity of domain C is uniquely sensitive to cysteine modification but that, as with gp32, ssDNA binding is not strictly dependent on zinc. The RPA heterotrimer is thus composed of at least four ssDNA binding domains and exhibits features of both bacterial and phage SSBs.

scholarly journals Replication protein A plays multifaceted roles complementary to specialized helicases in processing G-quadruplex DNA

iScience ◽  
2021 ◽  
pp. 102493
Author(s):  
Yi-Ran Wang ◽  
Ting-Ting Guo ◽  
Ya-Ting Zheng ◽  
Chang-Wei Lai ◽  
Bo Sun ◽  
...  
Keyword(s):  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Quadruplex Dna ◽  
G Quadruplex
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