scholarly journals Novel Checkpoint Response to Genotoxic Stress Mediated by Nucleolin-Replication Protein A Complex Formation

2005 ◽  
Vol 25 (6) ◽  
pp. 2463-2474 ◽  
Author(s):  
Kyung Kim ◽  
Diana D. Dimitrova ◽  
Kristine M. Carta ◽  
Anjana Saxena ◽  
Mariza Daras ◽  
...  
Keyword(s):  
Dna Replication ◽  
Complex Formation ◽  
Protein A ◽  
Resonance Energy ◽  
Genotoxic Stress ◽  
Replication Protein A ◽  
Replication Protein ◽  
Chromosomal Dna

ABSTRACT Human replication protein A (RPA), the primary single-stranded DNA-binding protein, was previously found to be inhibited after heat shock by complex formation with nucleolin. Here we show that nucleolin-RPA complex formation is stimulated after genotoxic stresses such as treatment with camptothecin or exposure to ionizing radiation. Complex formation in vitro and in vivo requires a 63-residue glycine-arginine-rich (GAR) domain located at the extreme C terminus of nucleolin, with this domain sufficient to inhibit DNA replication in vitro. Fluorescence resonance energy transfer studies demonstrate that the nucleolin-RPA interaction after stress occurs both in the nucleoplasm and in the nucleolus. Expression of the GAR domain or a nucleolin mutant (TM) with a constitutive interaction with RPA is sufficient to inhibit entry into S phase. Increasing cellular RPA levels by overexpression of the RPA2 subunit minimizes the inhibitory effects of nucleolin GAR or TM expression on chromosomal DNA replication. The arrest is independent of p53 activation by ATM or ATR and does not involve heightened expression of p21. Our data reveal a novel cellular mechanism that represses genomic replication in response to genotoxic stress by inhibition of an essential DNA replication factor.

scholarly journals Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers

2004 ◽  
Vol 24 (5) ◽  
pp. 1930-1943 ◽  
Author(s):  
Vitaly M. Vassin ◽  
Marc S. Wold ◽  
James A. Borowiec
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Protein A ◽  
Simian Virus 40 ◽  
Replication Protein A ◽  
Replication Protein ◽  
Wild Type ◽  
Chromosomal Dna

ABSTRACT Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2D) or alanine (RPA2A). Although RPA2D was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2D mutant was selectively unable to associate with replication centers in vivo. In cells containing greatly reduced levels of endogenous RPA2, RPA2D again did not localize to replication sites, indicating that the defect in supporting chromosomal DNA replication is not due to competition with the wild-type protein. Use of phosphospecific antibodies demonstrated that endogenous hyperphosphorylated RPA behaves similarly to RPA2D. In contrast, under DNA damage or replication stress conditions, RPA2D, like RPA2A and wild-type RPA2, was competent to associate with DNA damage foci as determined by colocalization with γ-H2AX. We conclude that RPA2 phosphorylation prevents RPA association with replication centers in vivo and potentially serves as a marker for sites of DNA damage.

scholarly journals Formation of a Complex between Nucleolin and Replication Protein a after Cell Stress Prevents Initiation of DNA Replication

2000 ◽  
Vol 149 (4) ◽  
pp. 799-810 ◽  
Author(s):  
Yaron Daniely ◽  
James A. Borowiec
Keyword(s):  
Dna Replication ◽  
Ribosome Biogenesis ◽  
Protein A ◽  
Simian Virus 40 ◽  
Replication Protein A ◽  
Cell Stress ◽  
Replication Protein ◽  
Binding Studies

We used a biochemical screen to identify nucleolin, a key factor in ribosome biogenesis, as a high-affinity binding partner for the heterotrimeric human replication protein A (hRPA). Binding studies in vitro demonstrated that the two proteins physically interact, with nucleolin using an unusual contact with the small hRPA subunit. Nucleolin significantly inhibited both simian virus 40 (SV-40) origin unwinding and SV-40 DNA replication in vitro, likely by nucleolin preventing hRPA from productive interaction with the SV-40 initiation complex. In vivo, use of epifluorescence and confocal microscopy showed that heat shock caused a dramatic redistribution of nucleolin from the nucleolus to the nucleoplasm. Nucleolin relocalization was concomitant with a tenfold increase in nucleolin–hRPA complex formation. The relocalized nucleolin significantly overlapped with the position of hRPA, but only poorly with sites of ongoing DNA synthesis. We suggest that the induced nucleolin–hRPA interaction signifies a novel mechanism that represses chromosomal replication after cell stress.

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 Analysis of a meiosis-specific URS1 site: sequence requirements and involvement of replication protein A.

1997 ◽  
Vol 17 (7) ◽  
pp. 3536-3546 ◽  
Author(s):  
V Gailus-Durner ◽  
C Chintamaneni ◽  
R Wilson ◽  
S J Brill ◽  
A K Vershon
Keyword(s):  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Sequence Specificity ◽  
Mobility Shift ◽  
Cell Extracts ◽  
Yeast Genes ◽  
Sequence Requirements

URS1 is a transcriptional repressor site found in the promoters of a wide variety of yeast genes that are induced under stress conditions. In the context of meiotic promoters, URS1 sites act as repressor sequences during mitosis and function as activator sites during meiosis. We have investigated the sequence requirements of the URS1 site of the meiosis-specific HOP1 gene (URS1H) and have found differences compared with a URS1 site from a nonmeiotic gene. We have also observed that the sequence specificity for meiotic activation at this site differs from that for mitotic repression. Base pairs flanking the conserved core sequence enhance meiotic induction but are not required for mitotic repression of HOP1. Electrophoretic mobility shift assays of mitotic and meiotic cell extracts show a complex pattern of DNA-protein complexes, suggesting that several different protein factors bind specifically to the site. We have determined that one of the complexes of URS1H is formed by replication protein A (RPA). Although RPA binds to the double-stranded URS1H site in vitro, it has much higher affinity for single-stranded than for double-stranded URS1H, and one-hybrid assays suggest that RPA does not bind to this site at detectable levels in vivo. In addition, conditional-lethal mutations in RPA were found to have no effect on URS1H-mediated repression. These results suggest that although RPA binds to URS1H in vitro, it does not appear to have a functional role in transcriptional repression through this site in vivo.

scholarly journals An interaction between the DNA repair factor XPA and replication protein A appears essential for nucleotide excision repair.

1995 ◽  
Vol 15 (10) ◽  
pp. 5396-5402 ◽  
Author(s):  
L Li ◽  
X Lu ◽  
C A Peterson ◽  
R J Legerski
Keyword(s):  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Protein A ◽  
Simian Virus 40 ◽  
Replication Protein A ◽  
Simian Virus ◽  
Replication Protein ◽  
Nucleotide Excision

Replication protein A (RPA) is required for simian virus 40-directed DNA replication in vitro and for nucleotide excision repair (NER). Here we report that RPA and the human repair protein XPA specifically interact both in vitro and in vivo. Mapping of the RPA-interactive domains in XPA revealed that both of the largest subunits of RPA, RPA-70 and RPA-34, interact with XPA at distinct sites. A domain involved in mediating the interaction with RPA-70 was located between XPA residues 153 and 176. Deletion of highly conserved motifs within this region identified two mutants that were deficient in binding RPA in vitro and highly defective in NER both in vitro and in vivo. A second domain mediating the interaction with RPA-34 was identified within the first 58 residues in XPA. Deletion of this region, however, only moderately affects the complementing activity of XPA in vivo. Finally, the XPA-RPA complex is shown to have a greater affinity for damaged DNA than XPA alone. Taken together, these results indicate that the interaction between XPA and RPA is required for NER but that only the interaction with RPA-70 is essential.

scholarly journals Replication protein A binds RNA and promotes R-loop formation

2020 ◽  
Vol 295 (41) ◽  
pp. 14203-14213
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 ◽  
Rna Metabolism ◽  
Replication Protein ◽  
Loop Formation ◽  
High Affinity ◽  
Replication Restart ◽  
Dna Replication Restart

Replication 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. To perform its functions, RPA binds ssDNA tightly. In contrast, it was presumed that RPA binds RNA weakly. However, recent data suggest that RPA may play a role in RNA metabolism. RPA stimulates RNA-templated DNA repair in vitro and associates in vivo with R-loops, the three-stranded structures consisting of an RNA-DNA hybrid and the displaced ssDNA strand. R-loops are common in the genomes of pro- and eukaryotes, including humans, and may play an important role in transcription-coupled homologous recombination and DNA replication restart. However, the mechanism of R-loop formation remains unknown. Here, we investigated the RNA-binding properties of human RPA and its possible role in R-loop formation. Using gel-retardation and RNA/DNA competition assays, we found that RPA binds RNA with an unexpectedly high affinity (KD ≈ 100 pm). Furthermore, RPA, by forming a complex with RNA, can promote R-loop formation with homologous dsDNA. In reconstitution experiments, we showed that human DNA polymerases can utilize RPA-generated R-loops for initiation of DNA synthesis, mimicking the process of replication restart in vivo. These results demonstrate that RPA binds RNA with high affinity, supporting the role of this protein in RNA metabolism and suggesting a mechanism of genome maintenance that depends on RPA-mediated DNA replication restart.

scholarly journals APOBEC1 cytosine deaminase activity on single-stranded DNA is suppressed by replication protein A

2020 ◽  
Author(s):  
Lai Wong ◽  
Frederick S Vizeacoumar ◽  
Franco J Vizeacoumar ◽  
Linda Chelico
Keyword(s):  
Genomic Dna ◽  
Cytidine Deaminase ◽  
Protein A ◽  
Cytosine Deaminase ◽  
Cancer Cell Line ◽  
Replication Protein A ◽  
Lung Cancer Cell Line ◽  
Replication Protein ◽  
Cancer Evolution

Abstract Many APOBEC cytidine deaminase members are known to induce ‘off-target’ cytidine deaminations in 5′TC motifs in genomic DNA that contribute to cancer evolution. In this report, we characterized APOBEC1, which is a possible cancer related APOBEC since APOBEC1 mRNA is highly expressed in certain types of tumors, such as lung adenocarcinoma. We found a low level of APOBEC1-induced DNA damage, as measured by γH2AX foci, in genomic DNA of a lung cancer cell line that correlated to its inability to compete in vitro with replication protein A (RPA) for ssDNA. This suggests that RPA can act as a defense against off-target deamination for some APOBEC enzymes. Overall, the data support the model that the ability of an APOBEC to compete with RPA can better predict genomic damage than combined analysis of mRNA expression levels in tumors and analysis of mutation signatures.

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