scholarly journals Replication protein A associates with nucleolar R loops and regulates rRNA transcription and nucleolar morphology

2021 ◽  
Author(s):  
Shuang Feng ◽  
James L. Manley
Keyword(s):  
Transcriptional Control ◽  
Protein A ◽  
Genotoxic Stress ◽  
Replication Protein A ◽  
Replication Protein ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Replication And Repair ◽  
Repair Factor ◽  
Rnap Ii

The nucleolus is an important cellular compartment in which ribosomal RNAs (rRNAs) are transcribed and where certain stress pathways that are crucial for cell growth are coordinated. Here we report novel functions of the DNA replication and repair factor replication protein A (RPA) in control of nucleolar homeostasis. We show that loss of the DNA:RNA helicase senataxin (SETX) promotes RPA nucleolar localization, and that this relocalization is dependent on the presence of R loops. Notably, this nucleolar RPA phenotype was also observed in the presence of camptothecin (CPT)-induced genotoxic stress, as well as in SETX-deficient AOA2 patient fibroblasts. Extending these results, we found that RPA is recruited to rDNA following CPT treatment, where RPA prevents R-loop-induced DNA double-strand breaks. Furthermore, we show that loss of RPA significantly decreased 47S pre-rRNA levels, which was accompanied by increased expression of both RNAP II-mediated “promoter and pre-rRNA antisense” RNA as well as RNAP I-transcribed intragenic spacer RNAs. Finally, and likely reflecting the above, we found that loss of RPA promoted nucleolar structural disorganization, characterized by the appearance of reduced size nucleoli. Our findings both indicate new roles for RPA in nucleoli through pre-rRNA transcriptional control and also emphasize that RPA function in nucleolar homeostasis is linked to R-loop resolution under both physiological and pathological conditions.

scholarly journals Chromatin-Directed Proteomics Identifies ZNF84 as a p53-Independent Regulator of p21 in Genotoxic Stress Response

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2115
Author(s):  
Anna Strzeszewska-Potyrała ◽  
Karolina Staniak ◽  
Joanna Czarnecka-Herok ◽  
Mahmoud-Reza Rafiee ◽  
Marcin Herok ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Genotoxic Stress ◽  
Vital Role ◽  
Dna Double Strand Breaks ◽  
Independent Manner ◽  
Strand Breaks ◽  
Knock Down ◽  
Proteomic Approach ◽  
Gene And Protein Expression ◽  
Transcriptomic Changes

The p21WAF1/Cip1 protein, encoded by CDKN1A, plays a vital role in senescence, and its transcriptional control by the tumour suppressor p53 is well-established. However, p21 can also be regulated in a p53-independent manner, by mechanisms that still remain less understood. We aimed to expand the knowledge about p53-independent senescence by looking for novel players involved in CDKN1A regulation. We used a chromatin-directed proteomic approach and identified ZNF84 as a novel regulator of p21 in various p53-deficient cell lines treated with cytostatic dose of doxorubicin. Knock-down of ZNF84, an as-yet un-characterized protein, inhibited p21 gene and protein expression in response to doxorubicin, it attenuated senescence and was associated with enhanced proliferation, indicating that ZNF84-deficiency can favor senescence bypass. ZNF84 deficiency was also associated with transcriptomic changes in genes governing various cancer-relevant processes e.g., mitosis. In cells with ZNF84 knock-down we discovered significantly lower level of H2AX Ser139 phosphorylation (γH2AX), which is triggered by DNA double strand breaks. Intriguingly, we observed a reverse correlation between the level of ZNF84 expression and survival rate of colon cancer patients. In conclusion, ZNF84, whose function was previously not recognized, was identified here as a critical p53-independent regulator of senescence, opening possibilities for its targeting in novel therapies of p53-null cancers.

The ionizing radiation-induced replication protein A phosphorylation response differs between ataxia telangiectasia and normal human cells

1993 ◽  
Vol 13 (12) ◽  
pp. 7222-7231
Author(s):  
V F Liu ◽  
D T Weaver
Keyword(s):  
Dna Repair ◽  
Ionizing Radiation ◽  
Gamma Irradiation ◽  
Ataxia Telangiectasia ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Inherited Disease ◽  
Dna Replication And Repair ◽  
In Cells

Replication protein A (RPA), the trimeric single-stranded DNA-binding protein complex of eukaryotic cells, is important to DNA replication and repair. Phosphorylation of the p34 subunit of RPA is modulated by the cell cycle, occurring during S and G2 but not during G1. The function of phosphorylated p34 remains unknown. We show that RPA p34 phosphorylation is significantly induced by ionizing radiation. The phosphorylated form, p36, is similar if not identical to the phosphorylated S/G2 form. gamma-Irradiation-induced phosphorylation occurs without new protein synthesis and in cells in G1. Mutation of cdc2-type protein kinase phosphorylation sites in p34 eliminates the ionizing radiation response. The gamma-irradiation-induced phosphorylation of RPA p34 is delayed in cells from ataxia telangiectasia, a human inherited disease conferring DNA repair defects and early-onset tumorigenesis. UV-induced phosphorylation of RPA p34 occurs less rapidly than gamma-irradiation-induced phosphorylation but is kinetically similar between ataxia telangiectasia and normal cells. This is the first time that modification of a repair protein, RPA, has been linked with a DNA damage response and suggests that phosphorylation may play a role in regulating DNA repair pathways.

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 Human RPA activates BLM’s bidirectional DNA unwinding from a nick

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Zhenheng Qin ◽  
Lulu Bi ◽  
Xi-Miao Hou ◽  
Siqi Zhang ◽  
Xia Zhang ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Single Molecule ◽  
Genome Stability ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
High Abundance ◽  
Dna Unwinding ◽  
Dna Replication And Repair

BLM is a multifunctional helicase that plays critical roles in maintaining genome stability. It processes distinct DNA substrates, but not nicked DNA, during many steps in DNA replication and repair. However, how BLM prepares itself for diverse functions remains elusive. Here, using a combined single-molecule approach, we find that a high abundance of BLMs can indeed unidirectionally unwind dsDNA from a nick when an external destabilizing force is applied. Strikingly, human replication protein A (hRPA) not only ensures that limited quantities of BLMs processively unwind nicked dsDNA under a reduced force but also permits the translocation of BLMs on both intact and nicked ssDNAs, resulting in a bidirectional unwinding mode. This activation necessitates BLM targeting on the nick and the presence of free hRPAs in solution whereas direct interactions between them are dispensable. Our findings present novel DNA unwinding activities of BLM that potentially facilitate its function switching in DNA repair.

scholarly journals Tipin-Replication Protein A Interaction Mediates Chk1 Phosphorylation by ATR in Response to Genotoxic Stress

2010 ◽  
Vol 285 (22) ◽  
pp. 16562-16571 ◽  
Author(s):  
Michael G. Kemp ◽  
Zafer Akan ◽  
Seçil Yilmaz ◽  
Mary Grillo ◽  
Stephanie L. Smith-Roe ◽  
...  
Keyword(s):  
Protein A ◽  
Genotoxic Stress ◽  
Replication Protein A ◽  
Replication Protein

scholarly journals The ionizing radiation-induced replication protein A phosphorylation response differs between ataxia telangiectasia and normal human cells.

1993 ◽  
Vol 13 (12) ◽  
pp. 7222-7231 ◽  
Author(s):  
V F Liu ◽  
D T Weaver
Keyword(s):  
Dna Repair ◽  
Ionizing Radiation ◽  
Gamma Irradiation ◽  
Ataxia Telangiectasia ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Inherited Disease ◽  
Dna Replication And Repair ◽  
In Cells

Replication protein A (RPA), the trimeric single-stranded DNA-binding protein complex of eukaryotic cells, is important to DNA replication and repair. Phosphorylation of the p34 subunit of RPA is modulated by the cell cycle, occurring during S and G2 but not during G1. The function of phosphorylated p34 remains unknown. We show that RPA p34 phosphorylation is significantly induced by ionizing radiation. The phosphorylated form, p36, is similar if not identical to the phosphorylated S/G2 form. gamma-Irradiation-induced phosphorylation occurs without new protein synthesis and in cells in G1. Mutation of cdc2-type protein kinase phosphorylation sites in p34 eliminates the ionizing radiation response. The gamma-irradiation-induced phosphorylation of RPA p34 is delayed in cells from ataxia telangiectasia, a human inherited disease conferring DNA repair defects and early-onset tumorigenesis. UV-induced phosphorylation of RPA p34 occurs less rapidly than gamma-irradiation-induced phosphorylation but is kinetically similar between ataxia telangiectasia and normal cells. This is the first time that modification of a repair protein, RPA, has been linked with a DNA damage response and suggests that phosphorylation may play a role in regulating DNA repair pathways.

scholarly journals A Proximity Ligation Method to Detect Proteins Bound to Single-Stranded DNA after DNA End Resection at DNA Double-Strand Breaks

2021 ◽  
Vol 5 (1) ◽  
pp. 3
Author(s):  
Faith C. Fowler ◽  
Jessica K. Tyler
Keyword(s):  
Homologous Recombination ◽  
Protein A ◽  
Replication Protein A ◽  
Strand Break ◽  
Replication Protein ◽  
Proximity Ligation Assay ◽  
Dna Double Strand Breaks ◽  
Color Channel ◽  
Single Stranded Dna ◽  
Proximity Ligation

After a DNA double-strand break, cells utilize either non-homologous end joining or homologous recombination to repair the broken DNA ends. Homologous recombination requires extensive nucleolytic processing of one of the DNA strands, resulting in long stretches of 3′ single-strand DNA overhangs. Typically, single-stranded DNA is measured using immunofluorescence microscopy to image the foci of replication protein A, a single-stranded DNA-binding protein. Microscopy analysis of bromodeoxyuridine foci under nondenaturing conditions has also been used to measure single-stranded DNA. Here, we describe a proximity ligation assay which uses genome-wide bromodeoxyuridine incorporation to label single-stranded DNA in order to measure the association of a protein of interest with single-stranded DNA. This method is advantageous over traditional foci analysis because it is more direct and specific than traditional foci co-localization microscopy methods, uses only one color channel, and can reveal protein-single-stranded DNA interactions that are rare and potentially undetectable using traditional microscopy methods. We show here the association of replication protein A and bromodeoxyuridine as proof-of-concept.

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