Novel homologs of replication protein A in Archaea: implications for the evolution of ssDNA-binding proteins

1998 ◽  
Vol 23 (8) ◽  
pp. 273-277 ◽  
Author(s):  
Frédéric Chédin ◽  
Erica M Seitz ◽  
Stephen C Kowalczykowski
Keyword(s):  
Binding Proteins ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Ssdna Binding ◽  
Ssdna Binding Proteins

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 Recruitment of Replication Protein A by the Papillomavirus E1 Protein and Modulation by Single-Stranded DNA

2004 ◽  
Vol 78 (4) ◽  
pp. 1605-1615 ◽  
Author(s):  
Yueh-Ming Loo ◽  
Thomas Melendy
Keyword(s):  
Dna Replication ◽  
Protein A ◽  
Replication Protein A ◽  
T Antigen ◽  
Replication Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Physical Interaction ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Papillomavirus Dna

ABSTRACT With the exception of viral proteins E1 and E2, papillomaviruses depend heavily on host replication machinery for replication of their viral genome. E1 and E2 are known to recruit many of the necessary cellular replication factors to the viral origin of replication. Previously, we reported a physical interaction between E1 and the major human single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA). E1 was determined to bind to the 70-kDa subunit of RPA, RPA70. In this study, using E1-affinity coprecipitation and enzyme-linked immunosorbent assay-based interaction assays, we show that E1 interacts with the major ssDNA-binding domain of RPA. Consistent with our previous report, no measurable interaction between E1 and the two smaller subunits of RPA was detected. The interaction of E1 with RPA was substantially inhibited by ssDNA. The extent of this inhibition was dependent on the length of the DNA. A 31-nucleotide (nt) oligonucleotide strongly inhibited the E1-RPA interaction, while a 16-nt oligonucleotide showed an intermediate level of inhibition. In contrast, a 10-nt oligonucleotide showed no observable effect on the E1-RPA interaction. This inhibition was not dependent on the sequence of the DNA. Furthermore, ssDNA also inhibited the interaction of RPA with papillomavirus E2, simian virus 40 T antigen, human polymerase alpha-primase, and p53. Taken together, our results suggest a potential role for ssDNA in modulating RPA-protein interactions, in particular, the RPA-E1 interactions during papillomavirus DNA replication. A model for recruitment of RPA by E1 during papillomavirus DNA replication is proposed.

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 binds RNA and promotes R-loop formation

2020 ◽  
Author(s):  
Olga M. Mazina ◽  
Srinivas Somarowthu ◽  
Lyudmila Y. Kadyrova ◽  
Andrey G. Baranovskiy ◽  
Tahir H. Tahirov ◽  
...  
Keyword(s):  
Dna Replication ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Loop Formation ◽  
Genome Maintenance ◽  
Ssdna Binding ◽  
Replication Restart ◽  
Ssdna Binding Protein

SUMMARYReplication protein A (RPA), a major eukaryotic ssDNA-binding protein, is essential for all metabolic processes that involve ssDNA including DNA replication, repair, and damage signaling. Surprisingly, we found here that RPA binds RNA in vitro with high affinity. Using native RIP method, we isolated RNA-RPA complexes from human cells. Furthermore, RPA promotes R-loop formation between RNA and homologous dsDNA. R-loops, the three-stranded nucleic acid structure consisting of an RNA-DNA hybrid and the displaced ssDNA strand, are common in human genome. R-loops may play an important role in transcription-coupled homologous recombination and DNA replication restart. We reconstituted the process of replication restart in vitro using RPA-generated R-loops and human DNA polymerases. These findings indicate that RPA may play a role in RNA metabolism and suggest a mechanism of genome maintenance that depends on RPA and RNA.

scholarly journals Determinants of replication protein A subunit interactions revealed using a phosphomimetic peptide

2020 ◽  
Vol 295 (52) ◽  
pp. 18449-18458
Author(s):  
Sungjin Lee ◽  
Jeongbeen Heo ◽  
Chin-Ju Park
Keyword(s):  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Domain Interaction ◽  
Docking Simulations ◽  
Ssdna Binding ◽  
Structural Details ◽  
Ssdna Binding Protein ◽  
A Subunit ◽  
Dna Resection

Replication protein A (RPA) is a eukaryotic ssDNA-binding protein and contains three subunits: RPA70, RPA32, and RPA14. Phosphorylation of the N-terminal region of the RPA32 subunit plays an essential role in DNA metabolism in processes such as replication and damage response. Phosphorylated RPA32 (pRPA32) binds to RPA70 and possibly regulates the transient RPA70-Bloom syndrome helicase (BLM) interaction to inhibit DNA resection. However, the structural details and determinants of the phosphorylated RPA32–RPA70 interaction are still unknown. In this study, we provide molecular details of the interaction between RPA70 and a mimic of phosphorylated RPA32 (pmRPA32) using fluorescence polarization and NMR analysis. We show that the N-terminal domain of RPA70 (RPA70N) specifically participates in pmRPA32 binding, whereas the unphosphorylated RPA32 does not bind to RPA70N. Our NMR data revealed that RPA70N binds pmRPA32 using a basic cleft region. We also show that at least 6 negatively charged residues of pmRPA32 are required for RPA70N binding. By introducing alanine mutations into hydrophobic positions of pmRPA32, we found potential points of contact between RPA70N and the N-terminal half of pmRPA32. We used this information to guide docking simulations that suggest the orientation of pmRPA32 in complex with RPA70N. Our study demonstrates detailed features of the domain-domain interaction between RPA70 and RPA32 upon phosphorylation. This result provides insight into how phosphorylation tunes transient bindings between RPA and its partners in DNA resection.

scholarly journals Human replication protein A binds single-stranded DNA in two distinct complexes.

1994 ◽  
Vol 14 (6) ◽  
pp. 3993-4001 ◽  
Author(s):  
L J Blackwell ◽  
J A Borowiec
Keyword(s):  
Protein A ◽  
Dna Recombination ◽  
Replication Protein A ◽  
Replication Protein ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Ssdna Binding Protein ◽  
Eukaryotic Dna ◽  
Gel Shift

Human replication protein A, a single-stranded DNA (ssDNA)-binding protein, is a required factor in eukaryotic DNA replication and DNA repair systems and has been suggested to function during DNA recombination. The protein is also a target of interaction for a variety of proteins that control replication, transcription, and cell growth. To understand the role of hRPA in these processes, we examined the binding of hRPA to defined ssDNA molecules. Employing gel shift assays that "titrated" the length of ssDNA, hRPA was found to form distinct multimeric complexes that could be detected by glutaraldehyde cross-linking. Within these complexes, monomers of hRPA utilized a minimum binding site size on ssDNA of 8 to 10 nucleotides (the hRPA8-10nt complex) and appeared to bind ssDNA cooperatively. Intriguingly, alteration of gel shift conditions revealed the formation of a second, distinctly different complex that bound ssDNA in roughly 30-nucleotide steps (the hRPA30nt complex), a complex similar to that described by Kim et al. (C. Kim, R. O. Snyder, and M. S. Wold, Mol. Cell. Biol. 12:3050-3059, 1992). Both the hRPA8-10nt and hRPA30nt complexes can coexist in solution. We speculate that the role of hRPA in DNA metabolism may be modulated through the ability of hRPA to bind ssDNA in these two modes.

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.

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